Methods, compositions, and kits using heterogeneous catalysts

ABSTRACT

Described herein are methods, compositions and kits utilizing heterogeneous metal catalysts for the preparation of cycloaddition compounds, such as triazoles and biomolecules.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 62/169,350, filed on Jun. 1, 2015, which is incorporatedby reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with the support of the United States governmentunder Contract number 1402029 by the National Science Foundation.

BACKGROUND OF THE INVENTION

Transition metal-catalyzed azide-alkyne cycloaddition is widely used inthe chemical modification of molecules and has demonstrated utility,particularly for bio-orthogonal conjugation reactions.

SUMMARY OF THE INVENTION

Described herein are methods, compositions, and kits for the preparationof cycloaddition compounds (e.g., triazole compounds from theazide-alkyne cycloaddition), including biomolecules, using aheterogeneous metal catalyst. The methods provided herein provide aconvenient syntheses and purification procedure using heterogeneousmetal catalysts.

In one aspect, provided herein is a method for preparing a cycloadditioncompound from a reaction catalyzed by a heterogeneous copper catalyst,comprising mixing an alkyne component and an azide component in anappropriate solvent to form a solution; transferring the solutioncontaining the alkyne component and the azide component to a column witha matrix barrier at the bottom of the column; wherein the columncomprises a matrix of a copper (I) catalyst; and passing the solutioncontaining the alkyne component and the azide component through thecolumn; wherein upon contact with the copper (I) catalyst within thematrix, the alkyne component and the azide component react to form thecycloaddition compound.

In another aspect, provided herein is a method for preparing acycloaddition compound from a reaction catalyzed by a heterogeneouscopper catalyst, comprising mixing an alkyne component and an azidecomponent in an appropriate solvent to form a solution; transferring thesolution containing the alkyne component and the azide component to acolumn with a matrix barrier at the bottom of the column; wherein thecolumn comprises a matrix of a copper (II) precatalyst and a reducingagent; wherein a copper (I) catalyst is generated from the reduction ofthe copper (II) precatalyst with the reducing agent; and passing thesolution containing the alkyne component and the azide component throughthe column; wherein upon contact with the copper (I) catalyst within thematrix, the alkyne component and the azide component react to form thecycloaddition compound.

In another aspect, provided herein is a method for preparing acycloaddition compound from a reaction catalyzed by a heterogeneouscopper catalyst, comprising mixing an alkyne component and an azidecomponent in an appropriate solvent to form a solution; transferring thesolution containing the alkyne component and the azide component to acolumn with a matrix barrier at the bottom of the column; wherein thecolumn comprises a matrix of a copper (II) precatalyst and zinc amalgam;wherein a copper (I) catalyst is generated from the reduction of thecopper (II) precatalyst with zinc amalgam; and passing the solutioncontaining the alkyne component and the azide component through thecolumn; wherein upon contact with the copper (I) catalyst within thematrix, the alkyne component and the azide component react to form thecycloaddition compound.

In some embodiments, the column is suitable use in gravity columnchromatography or centrifugal column chromatography. In someembodiments, passing the solution containing the alkyne component andthe azide component through the column is through gravity. In certainembodiments, passing the solution containing the alkyne component andthe azide component through the column further comprises the use ofpositive pressure from air or a compressed gas. In some embodiments,passing the solution containing the alkyne component and the azidecomponent through the column is through centrifuging the column for asufficient period of time.

In another aspect, provided herein is a method for preparing acycloaddition compound from a reaction catalyzed by a heterogeneouscopper catalyst, comprising mixing an alkyne component and an azidecomponent in an appropriate solvent to form a solution; transferring thesolution containing the alkyne component and the azide component to acentrifuge column with a matrix barrier at the bottom of the centrifugecolumn; wherein the centrifuge column comprises a matrix of a copper (I)catalyst; and passing the solution containing the alkyne component andthe azide component through the centrifuge column by centrifuging thecentrifuge column for a sufficient period of time; wherein upon contactwith the copper (I) catalyst within the matrix, the alkyne component andthe azide component react to form the cycloaddition compound.

In another aspect, provided herein is a method for preparing acycloaddition compound from a reaction catalyzed by a heterogeneouscopper catalyst, comprising mixing an alkyne component and an azidecomponent in an appropriate solvent to form a solution; transferring thesolution containing the alkyne component and the azide component to acentrifuge column with a matrix barrier at the bottom of the centrifugecolumn; wherein the centrifuge column comprises a matrix of a copper(II) precatalyst and a reducing agent; wherein a copper (I) catalyst isgenerated from the reduction of the copper (II) precatalyst with thereducing agent; and passing the solution containing the alkyne componentand the azide component through centrifuge column by centrifuging thecentrifuge column for a sufficient period of time; wherein upon contactwith the copper (I) catalyst within the matrix, the alkyne component andthe azide component react to form the cycloaddition compound.

In another aspect, provided herein is a method for preparing acycloaddition compound from a reaction catalyzed by a heterogeneouscopper catalyst, comprising mixing an alkyne component and an azidecomponent in an appropriate solvent to form a solution; transferring thesolution containing the alkyne component and the azide component to acentrifuge column with a matrix barrier at the bottom of the centrifugecolumn; wherein the centrifuge column comprises a matrix of a copper(II) precatalyst and zinc amalgam; wherein a copper (I) catalyst isgenerated from the reduction of the copper (II) precatalyst with zincamalgam; and passing the solution containing the alkyne component andthe azide component through the centrifuge column by centrifuging thecentrifuge column for a sufficient period of time; wherein upon contactwith the copper (I) catalyst within the matrix, the alkyne component andthe azide component react to form the cycloaddition compound.

In another aspect, provided herein is a method for preparing acycloaddition compound from a reaction catalyzed by a heterogeneouscopper catalyst, comprising mixing an alkyne component and an azidecomponent in an appropriate solvent to form a solution; transferring thesolution containing the alkyne component and the azide component to agravity column with a matrix barrier at the bottom of the gravitycolumn; wherein the gravity column comprises a matrix of a copper (I)catalyst; and passing the solution containing the alkyne component andthe azide component through the gravity column; wherein upon contactwith the copper (I) catalyst within the matrix, the alkyne component andthe azide component react to form the cycloaddition compound.

In another aspect, provided herein is a method for preparing acycloaddition compound from a reaction catalyzed by a heterogeneouscopper catalyst, comprising mixing an alkyne component and an azidecomponent in an appropriate solvent to form a solution; transferring thesolution containing the alkyne component and the azide component to agravity column with a matrix barrier at the bottom of the gravitycolumn; wherein the gravity column comprises a matrix of a copper (II)precatalyst and a reducing agent; wherein a copper (I) catalyst isgenerated from the reduction of the copper (II) precatalyst with thereducing agent; and passing the solution containing the alkyne componentand the azide component through the gravity column; wherein upon contactwith the copper (I) catalyst within the matrix, the alkyne component andthe azide component react to form the cycloaddition compound.

In another aspect, provided herein is a method for preparing acycloaddition compound from a reaction catalyzed by a heterogeneouscopper catalyst, comprising mixing an alkyne component and an azidecomponent in an appropriate solvent to form a solution; transferring thesolution containing the alkyne component and the azide component to agravity column with a matrix barrier at the bottom of the gravitycolumn; wherein the gravity column comprises a matrix of a copper (II)precatalyst and zinc amalgam; wherein a copper (I) catalyst is generatedfrom the reduction of the copper (II) precatalyst with zinc amalgam; andpassing the solution containing the alkyne component and the azidecomponent through the gravity column; wherein upon contact with thecopper (I) catalyst within the matrix, the alkyne component and theazide component react to form the cycloaddition compound.

In some embodiments, the alkyne component and/or the azide componentcomprises a small molecule, a protein, a peptide, an amino acid, anoligonucleotide, a nucleotide, a nucleoside, a carbohydrate, or afluorophore. In some embodiments, the alkyne component comprises a smallmolecule, a protein, a peptide, an amino acid, an oligonucleotide, anucleotide, a nucleoside, a carbohydrate, or a fluorophore. In someembodiments, the azide component comprises a small molecule, a protein,a peptide, an amino acid, an oligonucleotide, a nucleotide, anucleoside, a carbohydrate, or a fluorophore. In some embodiments, thealkyne component comprises an oligonucleotide and the azide componentcomprises a carbohydrate. In some embodiments, the alkyne componentcomprises a peptide and the azide component comprises a fluorophore. Insome embodiments, the alkyne component and/or the azide componentcontains cells and/or cell lysates. In some embodiments, the alkynecomponent and/or the azide component contains cells. In someembodiments, the alkyne component and/or the azide component containscell lysates. In some embodiments, the alkyne component contains cells.In some embodiments, the azide component contains cells. In someembodiments, the alkyne component contains cell lysates. In someembodiments, the azide component contains cell lysates.

In another aspect, provided herein is a method for preparing aglycosylated DNA from a reaction catalyzed by a heterogeneous coppercatalyst, comprising mixing an alkynyl DNA component and an azidocarbohydrate component in an appropriate solvent to form a solution;transferring the solution containing the alkynyl DNA component and theazido carbohydrate component to a centrifuge column with a matrixbarrier at the bottom of the centrifuge column; wherein the centrifugecolumn comprises a matrix of a copper (II) precatalyst and a reducingagent; wherein a copper (I) catalyst is generated from the reduction ofthe copper (II) precatalyst with the reducing agent; and passing thesolution containing the alkynyl DNA component and the azido carbohydratecomponent through the centrifuge column by centrifuging the centrifugecolumn for a sufficient period of time; wherein upon contact with thecopper (I) catalyst within the matrix, the alkynyl DNA component and theazido carbohydrate component react to form a glycosylated DNA.

In another aspect, provided herein is a method for preparing afluorescent labeled peptide from a reaction catalyzed by a heterogeneouscopper catalyst, comprising mixing an alkynyl peptide component and anazido fluorogenic dye component in an appropriate solvent to form asolution; transferring the solution containing the alkynyl peptidecomponent and the azido fluorogenic dye component to a centrifuge columnwith a matrix barrier at the bottom of the centrifuge column; whereinthe centrifuge column comprises a matrix of copper (II) precatalyst anda reducing agent; wherein a copper (I) catalyst is generated from thereduction of the copper (II) precatalyst with the reducing agent; andpassing the solution containing the alkynyl peptide component and theazido fluorogenic dye component through the centrifuge column bycentrifuging the centrifuge column for a sufficient period of time;wherein upon contact with the copper (I) catalyst within the matrix, thealkynyl peptide component and the azido fluorogenic component react toform a fluorescent labeled peptide.

In some embodiments, the cycloaddition compound, glycosylated DNA, orfluorescent labeled peptide is eluted with an appropriate buffersolution followed by centrifuging the centrifuge column for a sufficientperiod of time. In some embodiments, the cycloaddition compound iseluted with an appropriate buffer solution followed by centrifuging thecentrifuge column for a sufficient period of time. In some embodiments,glycosylated DNA is eluted with an appropriate buffer solution followedby centrifuging the centrifuge column for a sufficient period of time.In some embodiments, the fluorescent labeled peptide is eluted with anappropriate buffer solution followed by centrifuging the centrifugecolumn for a sufficient period of time.

In some embodiments, the cycloaddition compound, glycosylated DNA, orfluorescent labeled peptide is a 1,2,3-triazole. In some embodiments,the cycloaddition compound is a 1,2,3-triazole. In some embodiments, theglycosylated DNA is a 1,2,3-triazole. In some embodiments, thefluorescent labeled peptide is a 1,2,3-triazole.

In some embodiments, the cycloaddition compound, glycosylated DNA, orfluorescent labeled peptide is a cell lysate derivatized product. Insome embodiments, the cell lysate derivatized product is free orsubstantially free of impurities derived from cells and/or cell lysates.In some embodiments, the impurities include but are not limited toproteins and related materials derived from cells and/or cell lysatesthat are able to pass through the column matrix. In some embodiments,the cell lysate derivatized product requires secondary purification toremove trace impurities derived from cells and/or cell lysates. In someembodiments, the cell lysate derivatized product is free orsubstantially free of impurities derived from cell lysates. In someembodiments, the impurities include but are not limited to proteins andrelated materials derived from cell lysates that are able to passthrough the column matrix. In some embodiments, the cell lysatederivatized product requires secondary purification to remove traceimpurities derived from cell lysates. In some embodiments, secondarypurification is any one of the methods disclosed herein for additionalpurification.

In some embodiments, the method further comprises passing the solutioncontaining the cycloaddition compound, glycosylated DNA, or fluorescentlabeled peptide through an additional matrix. In some embodiments, themethod further comprises passing the solution containing thecycloaddition compound through an additional matrix. In someembodiments, the method further comprises passing the solutioncontaining the glycosylated DNA through an additional matrix. In someembodiments, the method further comprises passing the solutioncontaining the fluorescent labeled peptide through an additional matrix.

In certain embodiments, the additional matrix is a resin suitable forthe purification of the cycloaddition compound, glycosylated DNA orfluorescent labeled peptide. In certain embodiments, the additionalmatrix is a resin suitable for the purification of the cycloadditioncompound. In certain embodiments, the additional matrix is a resinsuitable for the purification of the glycosylated DNA. In certainembodiments, the additional matrix is a resin suitable for thepurification of the fluorescent labeled peptide. In certain embodiments,the additional matrix is a size-exclusion resin.

In some embodiments, the method further comprises passing the solutioncontaining the cycloaddition compound, glycosylated DNA, or fluorescentlabeled peptide through an additional matrix that is a resin suitablefor the purification of the cycloaddition compound, glycosylated DNA, orfluorescent labeled peptide. In some embodiments, the method furthercomprises passing the solution containing the cycloaddition compoundthrough an additional matrix that is a resin suitable for thepurification of the cycloaddition compound. In some embodiments, themethod further comprises passing the solution containing theglycosylated DNA through an additional matrix that is a resin suitablefor the purification of the glycosylated DNA. In some embodiments, themethod further comprises passing the solution containing the fluorescentlabeled peptide through an additional matrix that is a resin suitablefor the purification of the fluorescent labeled peptide. In certainembodiments, the additional matrix is a size-exclusion resin.

In some embodiments, the matrix further comprises an ion-exchange resin.In certain embodiments, the ion-exchange resin is an anion exchangeresin or a cation exchange resin. In some embodiments, the column,centrifuge column, or gravity column further comprises a size-exclusionresin. In some embodiments, the column further comprises asize-exclusion resin. In some embodiments, the centrifuge column furthercomprises a size-exclusion resin. In some embodiments, the gravitycolumn further comprises a size-exclusion resin.

In some embodiments, the copper (II) precatalyst further comprises aligand. In some embodiments, the reducing agent is zinc amalgam. In someembodiments, the method is suitable for use in microspin columnchromatography.

In one aspect, provided herein is a method for preparing a cell lysatederivatized product from a reaction catalyzed by a heterogeneous coppercatalyst, comprising mixing an alkyne component and an azide componentin an appropriate solvent to form a solution; transferring the solutioncontaining the alkyne component and the azide component to a column witha matrix barrier at the bottom of the column; wherein the columncomprises a matrix of a copper (I) catalyst; and passing the solutioncontaining the alkyne component and the azide component through thecolumn; wherein upon contact with the copper (I) catalyst within thematrix, the alkyne component and the azide component react to form thecell lysate derivatized product.

In another aspect, provided herein is a method for preparing a celllysate derivatized product from a reaction catalyzed by a heterogeneouscopper catalyst, comprising mixing an alkyne component and an azidecomponent in an appropriate solvent to form a solution; transferring thesolution containing the alkyne component and the azide component to acolumn with a matrix barrier at the bottom of the column; wherein thecolumn comprises a matrix of a copper (II) precatalyst and a reducingagent; wherein a copper (I) catalyst is generated from the reduction ofthe copper (II) precatalyst with the reducing agent; and passing thesolution containing the alkyne component and the azide component throughthe column; wherein upon contact with the copper (I) catalyst within thematrix, the alkyne component and the azide component react to form thecell lysate derivatized product.

In another aspect, provided herein is a method for preparing a celllysate derivatized product from a reaction catalyzed by a heterogeneouscopper catalyst, comprising mixing an alkyne component and an azidecomponent in an appropriate solvent to form a solution; transferring thesolution containing the alkyne component and the azide component to acolumn with a matrix barrier at the bottom of the column; wherein thecolumn comprises a matrix of a copper (II) precatalyst and zinc amalgam;wherein a copper (I) catalyst is generated from the reduction of thecopper (II) precatalyst with zinc amalgam; and passing the solutioncontaining the alkyne component and the azide component through thecolumn; wherein upon contact with the copper (I) catalyst within thematrix, the alkyne component and the azide component react to form thecell lysate derivatized product.

In some embodiments, the column is suitable use in gravity columnchromatography or centrifugal column chromatography. In someembodiments, passing the solution containing the alkyne component andthe azide component through the column is through gravity. In certainembodiments, passing the solution containing the alkyne component andthe azide component through the column further comprises the use ofpositive pressure from air or a compressed gas. In some embodiments,passing the solution containing the alkyne component and the azidecomponent through the column is through centrifuging the column for asufficient period of time.

In another aspect, provided herein is a method for preparing a celllysate derivatized product from a reaction catalyzed by a heterogeneouscopper catalyst, comprising mixing an alkyne component and an azidecomponent in an appropriate solvent to form a solution; transferring thesolution containing the alkyne component and the azide component to acentrifuge column with a matrix barrier at the bottom of the centrifugecolumn; wherein the centrifuge column comprises a matrix of a copper (I)catalyst; and passing the solution containing the alkyne component andthe azide component through the centrifuge column by centrifuging thecentrifuge column for a sufficient period of time; wherein upon contactwith the copper (I) catalyst within the matrix, the alkyne component andthe azide component react to form the cell lysate derivatized product.

In another aspect, provided herein is a method for preparing a celllysate derivatized product from a reaction catalyzed by a heterogeneouscopper catalyst, comprising mixing an alkyne component and an azidecomponent in an appropriate solvent to form a solution; transferring thesolution containing the alkyne component and the azide component to acentrifuge column with a matrix barrier at the bottom of the centrifugecolumn; wherein the centrifuge column comprises a matrix of a copper(II) precatalyst and a reducing agent; wherein a copper (I) catalyst isgenerated from the reduction of the copper (II) precatalyst with thereducing agent; and passing the solution containing the alkyne componentand the azide component through centrifuge column by centrifuging thecentrifuge column for a sufficient period of time; wherein upon contactwith the copper (I) catalyst within the matrix, the alkyne component andthe azide component react to form the cell lysate derivatized product.

In another aspect, provided herein is a method for preparing a celllysate derivatized product from a reaction catalyzed by a heterogeneouscopper catalyst, comprising mixing an alkyne component and an azidecomponent in an appropriate solvent to form a solution; transferring thesolution containing the alkyne component and the azide component to acentrifuge column with a matrix barrier at the bottom of the centrifugecolumn; wherein the centrifuge column comprises a matrix of a copper(II) precatalyst and zinc amalgam; wherein a copper (I) catalyst isgenerated from the reduction of the copper (II) precatalyst with zincamalgam; and passing the solution containing the alkyne component andthe azide component through the centrifuge column by centrifuging thecentrifuge column for a sufficient period of time; wherein upon contactwith the copper (I) catalyst within the matrix, the alkyne component andthe azide component react to form the cell lysate derivatized product.

In another aspect, provided herein is a method for preparing a celllysate derivatized product from a reaction catalyzed by a heterogeneouscopper catalyst, comprising mixing an alkyne component and an azidecomponent in an appropriate solvent to form a solution; transferring thesolution containing the alkyne component and the azide component to agravity column with a matrix barrier at the bottom of the gravitycolumn; wherein the gravity column comprises a matrix of a copper (I)catalyst; and passing the solution containing the alkyne component andthe azide component through the gravity column; wherein upon contactwith the copper (I) catalyst within the matrix, the alkyne component andthe azide component react to form the cell lysate derivatized product.

In another aspect, provided herein is a method for preparing a celllysate derivatized product from a reaction catalyzed by a heterogeneouscopper catalyst, comprising mixing an alkyne component and an azidecomponent in an appropriate solvent to form a solution; transferring thesolution containing the alkyne component and the azide component to agravity column with a matrix barrier at the bottom of the gravitycolumn; wherein the gravity column comprises a matrix of a copper (II)precatalyst and a reducing agent; wherein a copper (I) catalyst isgenerated from the reduction of the copper (II) precatalyst with thereducing agent; and passing the solution containing the alkyne componentand the azide component through the gravity column; wherein upon contactwith the copper (I) catalyst within the matrix, the alkyne component andthe azide component react to form the cell lysate derivatized product.

In another aspect, provided herein is a method for preparing a celllysate derivatized product from a reaction catalyzed by a heterogeneouscopper catalyst, comprising mixing an alkyne component and an azidecomponent in an appropriate solvent to form a solution; transferring thesolution containing the alkyne component and the azide component to agravity column with a matrix barrier at the bottom of the gravitycolumn; wherein the gravity column comprises a matrix of a copper (II)precatalyst and zinc amalgam; wherein a copper (I) catalyst is generatedfrom the reduction of the copper (II) precatalyst with zinc amalgam; andpassing the solution containing the alkyne component and the azidecomponent through the gravity column; wherein upon contact with thecopper (I) catalyst within the matrix, the alkyne component and theazide component react to form the cell lysate derivatized product.

In some embodiments, the alkyne component and/or the azide componentcomprises a small molecule, a protein, a peptide, an amino acid, anoligonucleotide, a nucleotide, a nucleoside, a carbohydrate, or afluorophore. In some embodiments, the alkyne component comprises a smallmolecule, a protein, a peptide, an amino acid, an oligonucleotide, anucleotide, a nucleoside, a carbohydrate, or a fluorophore. In someembodiments, the azide component comprises a small molecule, a protein,a peptide, an amino acid, an oligonucleotide, a nucleotide, anucleoside, a carbohydrate, or a fluorophore. In some embodiments, thealkyne component comprises an oligonucleotide and the azide componentcomprises a carbohydrate. In some embodiments, the alkyne componentcomprises a peptide and the azide component comprises a fluorophore. Insome embodiments, the alkyne component and/or the azide componentcontains cells and/or cell lysates. In some embodiments, the alkynecomponent and/or the azide component contains cells. In someembodiments, the alkyne component and/or the azide component containscell lysates. In some embodiments, the alkyne component contains cells.In some embodiments, the azide component contains cells. In someembodiments, the alkyne component contains cell lysates. In someembodiments, the azide component contains cell lysates.

In some embodiments, the cell lysate derivatized product is eluted withan appropriate buffer solution followed by centrifuging the centrifugecolumn for a sufficient period of time. In some embodiments, the celllysate derivatized product is eluted with an appropriate buffer solutionfollowed by centrifuging the centrifuge column for a sufficient periodof time.

In some embodiments, the cell lysate derivatized product is a1,2,3-triazole. In some embodiments, the cell lysate derivatized productis free or substantially free of impurities derived from cells and/orcell lysates. In some embodiments, the impurities include but are notlimited to proteins and related materials derived from cells and/or celllysates that are able to pass through the column matrix. In someembodiments, the cell lysate derivatized product requires secondarypurification to remove trace impurities derived from cells and/or celllysates. In some embodiments, the cell lysate derivatized product isfree or substantially free of impurities derived from cell lysates. Insome embodiments, the impurities include but are not limited to proteinsand related materials derived from cell lysates that are able to passthrough the column matrix. In some embodiments, the cell lysatederivatized product requires secondary purification to remove traceimpurities derived from cell lysates. In some embodiments, secondarypurification is any one of the methods disclosed herein for additionalpurification.

In some embodiments, the method further comprises passing the solutioncontaining the cell lysate derivatized product through an additionalmatrix. In certain embodiments, the additional matrix is a resinsuitable for the purification of the cell lysate derivatized product. Incertain embodiments, the additional matrix is a size-exclusion resin.

In some embodiments, the method further comprises passing the solutioncontaining the cell lysate derivatized product through an additionalmatrix that is a resin suitable for the purification of the cell lysatederivatized product. In certain embodiments, the additional matrix is asize-exclusion resin.

In some embodiments, the matrix further comprises an ion-exchange resin.In certain embodiments, the ion-exchange resin is an anion exchangeresin or a cation exchange resin. In some embodiments, the column,centrifuge column, or gravity column further comprises a size-exclusionresin. In some embodiments, the column further comprises asize-exclusion resin. In some embodiments, the centrifuge column furthercomprises a size-exclusion resin. In some embodiments, the gravitycolumn further comprises a size-exclusion resin.

In some embodiments, the copper (II) precatalyst further comprises aligand. In some embodiments, the reducing agent is zinc amalgam. In someembodiments, the method is suitable for use in microspin columnchromatography.

In another aspect, provided herein is a column matrix comprising aheterogeneous metal catalyst. In another aspect, provided herein is acolumn matrix comprising a heterogeneous metal catalyst that catalyzesthe azide-alkyne cycloaddition.

In some embodiments, the column matrix is suitable for catalyzing anazide-alkyne cycloaddition. In some embodiments, the heterogeneous metalcatalyst comprises a copper catalyst, a ruthenium catalyst, a silvercatalyst, or a zinc catalyst. In some embodiments, the heterogeneousmetal catalyst comprises a copper catalyst. In some embodiments, theheterogeneous metal catalyst comprises a ruthenium catalyst. In someembodiments, the heterogeneous metal catalyst comprises a silvercatalyst. In some embodiments, the heterogeneous metal comprises a zinccatalyst.

In some embodiments, the copper catalyst further comprises a ligand. Insome embodiments, the copper catalyst comprises a copper (II)precatalyst and reducing agent. In some embodiments, the copper (II)precatalyst further comprises a ligand. In some embodiments, thereducing agent is zinc amalgam.

In another aspect, provided herein is a column matrix comprising aheterogeneous copper catalyst. In another aspect, provided herein is acolumn matrix comprising a heterogeneous copper catalyst that catalyzesthe azide-alkyne cycloaddition.

In some embodiments, the heterogeneous copper catalyst further comprisesa ligand. In some embodiments, the heterogeneous copper catalystcomprises a copper (II) precatalyst and reducing agent. In someembodiments, the copper (II) precatalyst further comprises a ligand. Insome embodiments, the reducing agent is zinc amalgam.

In another aspect, provided herein is a column matrix comprising acopper (I) catalyst. In another aspect, provided herein is a columnmatrix comprising a copper (II) precatalyst and a reducing agent. Inanother aspect, provided herein is a column matrix comprising a copper(II) precatalyst and zinc amalgam.

In another aspect, provided herein is a column matrix for use inbioconjugation comprising a copper (I) catalyst. In another aspect,provided herein is a column matrix for use in bioconjugation comprisinga copper (II) precatalyst and a reducing agent. In another aspect,provided herein is a column matrix for use in bioconjugation comprisinga copper (II) precatalyst and zinc amalgam. In another aspect, providedherein is a column matrix for use in bioconjugation catalyzed by aheterogeneous copper catalyst comprising a copper (I) catalyst. Inanother aspect, provided herein is a column matrix for use inbioconjugation catalyzed by a heterogeneous copper comprising a copper(II) precatalyst and a reducing agent. In another aspect, providedherein is a column matrix for use in bioconjugation catalyzed by aheterogeneous copper comprising a copper (II) precatalyst and zincamalgam.

In some embodiments, the column matrix further comprises an ion-exchangeresin. In certain embodiments, the ion-exchange resin is an anionexchange resin or a cation exchange resin. In some embodiments, thecolumn matrix further comprises a size-exclusion resin. In someembodiments, the column matrix further comprises an additional columnmatrix. In certain embodiments, the additional column matrix is a resinsuitable for the purification of a cycloaddition compound formed byazide-alkyne cycloaddition. In certain embodiments, the additionalcolumn matrix is a resin suitable for the purification of a product. Incertain embodiments, the product is a triazole formed from aheterogeneous metal-catalyzed azide alkyne cycloaddition. In certainembodiments, the additional column matrix is a size-exclusion resin.

In some embodiments, the column matrix further comprises an additionalcolumn matrix that is a resin that is suitable for the purification of aproduct. In some embodiments, the column matrix further comprises anadditional column matrix that is a resin that is suitable for thepurification of a product formed from the azide-alkyne cycloaddition. Incertain embodiments, the additional column matrix is a size-exclusionresin.

In some embodiments, the copper (II) precatalyst further comprises aligand. In some embodiments, the reducing agent is zinc amalgam. In someembodiments, the column matrix is suitable for use in gravity columnchromatography or in centrifugal column chromatography. In someembodiments, the column matrix is suitable for use in gravity columnchromatography. In some embodiments, the column matrix is suitable foruse in centrifugal column chromatography. In certain embodiments, thecolumn matrix is suitable for use in microspin column chromatography. Insome embodiments, the column matrix is suitable for use in azide-alkynecycloaddition.

In another aspect, provided herein is a column comprising a matrix of aheterogeneous metal catalyst. In another aspect, provided herein is acolumn comprising a matrix of a heterogeneous metal catalyst thatcatalyzes the azide-alkyne cycloaddition.

In some embodiments, the heterogeneous metal catalyst comprises a coppercatalyst, a ruthenium catalyst, a silver catalyst, or a zinc catalyst.In some embodiments, the heterogeneous metal catalyst comprises a coppercatalyst. In some embodiments, the heterogeneous metal catalystcomprises a ruthenium catalyst. In some embodiments, the heterogeneousmetal catalyst comprises a silver catalyst. In some embodiments, theheterogeneous metal comprises a zinc catalyst.

In some embodiments, the copper catalyst further comprises a ligand. Insome embodiments, the copper catalyst comprises a copper (II)precatalyst and a reducing agent. In some embodiments, the copper (II)precatalyst further comprises a ligand. In some embodiments, thereducing agent is zinc amalgam.

In another aspect, provided herein is a column comprising a matrix of aheterogeneous copper catalyst. In another aspect, provided herein is acolumn comprising a matrix of a heterogeneous copper catalyst thatcatalyzes the azide-alkyne cycloaddition.

In some embodiments, the heterogeneous copper catalyst further comprisesa ligand. In some embodiments, the heterogeneous copper catalystcomprises a copper (II) precatalyst and a reducing agent. In someembodiments, the copper (II) precatalyst further comprises a ligand. Insome embodiments, the reducing agent is zinc amalgam.

In another aspect, provided herein is a column comprising a matrix of acopper (I) catalyst. In another aspect, provided herein is a columncomprising a matrix of a copper (II) precatalyst and a reducing agent.In another aspect, provided herein is a column comprising a matrix of acopper (II) precatalyst and zinc amalgam.

In another aspect, provided herein is a column for use in bioconjugationcomprising a matrix of a copper (I) catalyst. In another aspect,provided herein is a column for use in bioconjugation comprising amatrix of a copper (II) precatalyst and a reducing agent. In anotheraspect, provided herein is a column for use in bioconjugation comprisinga matrix of a copper (II) precatalyst and zinc amalgam. In anotheraspect, provided herein is a column for use in bioconjugation catalyzedby a heterogeneous copper catalyst comprising a matrix of a copper (I)catalyst. In another aspect, provided herein is a column for use inbioconjugation catalyzed by a heterogeneous copper catalyst comprising amatrix of a copper (II) precatalyst and a reducing agent. In anotheraspect, provided herein is a column for use in bioconjugation catalyzedby a heterogeneous copper catalyst comprising a matrix of a copper (II)precatalyst and zinc amalgam.

In some embodiments, the matrix further comprises an ion-exchange resin.In some embodiments, the ion-exchange resin is an anion exchange resinor a cation exchange resin. In some embodiments, the matrix furthercomprises a size-exclusion resin. In some embodiments, the columnfurther comprises an additional matrix. In certain embodiments, theadditional matrix is a resin suitable for the purification of acycloaddition compound formed from an azide-alkyne cycloaddition. Incertain embodiments, the additional matrix is a resin suitable for thepurification of a product. In certain embodiments, the product is atriazole formed from a heterogeneous metal-catalyzed azide-alkynecycloaddition. In certain embodiments, the additional matrix is asize-exclusion resin.

In some embodiments, the column further comprises an additional matrixthat is a resin suitable for the purification of a cycloadditioncompound formed from the azide-alkyne cycloaddition.

In some embodiments, the copper (II) precatalyst further comprises aligand. In some embodiments, the reducing agent is zinc amalgam. In someembodiments, the column is a gravity column or a centrifuge column. Incertain embodiments, the column is a centrifuge column. In certainembodiments, the column is a gravity column. In some embodiments, thecentrifuge column is suitable for use in microspin columnchromatography. In some embodiments, the column is suitable for use inazide-alkyne cycloaddition.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts a schematic diagram of a particular embodiment of thecolumn, wherein the column comprises a matrix comprising a heterogeneousmetal catalyst and the column further comprises an additional matrix.

FIG. 2 depicts a schematic diagram of a particular embodiment of thecentrifuge column, wherein the centrifuge column comprises a matrixcontaining a heterogeneous copper catalyst (II) precatalyst and reducingagent, and the centrifuge column further comprises a size-exclusionresin.

FIG. 3A and FIG. 3B depict the HPLC traces for crude product followingDNA-ethynyl/azido-glucose conjugation via centrifuge column, or spincolumn, (FIG. 3A) and conventional solution-phase coupling using sodiumascorbate as the reducing agent (FIG. 3B).

FIG. 4A and FIG. 4B depict the HPLC traces for crude product following8-Arg/coumarin conjugation via centrifuge column, or spin column, (FIG.4A), and conventional solution-phase coupling using sodium ascorbate asthe reducing agent (FIG. 4B). (*indicates 8-Arg unmodified with anethynyl group)

FIG. 5 depicts the calibration curve obtained for the ICP-AES analysisof Cu in spin column flow-through loaded with Cu-ligand catalyst.

DETAILED DESCRIPTION OF THE INVENTION

An emerging field has been in developing azide-alkyne cycloadditionprocesses that use heterogeneous metal catalysts as the advantages ofheterogeneous metal systems include easy product separation, minimalwaste generation during catalyst separation, catalyst recyclability, andfacile catalyst removal. Described herein are methods, compositions andkits utilizing heterogeneous metal catalysts, in particular coppercatalysts, for the preparation of compounds, such as triazoles from anazide-alkyne cycloaddition and includes biomolecules.

“Click chemistry” reactions are a set of highly reliable and selectivereactions that allow for the rapid synthesis of new compounds. One ofthe most well-known click reactions is the Huisgen [3+2] azide-alkynecycloaddition. This reaction has the following advantages: the reactionis generally high yielding and robust; the alkyne and azide componentsare incorporated into a wide range of substrates; and the triazoleformed is essentially chemically inert to reactive conditions.Furthermore, as the azide moiety is not present in naturally existingcompounds, the application of the azide-alkyne cycloaddition is ofparticular interest in bioconjugation.

In the absence of a catalyst, the Huisgen [3+2] azide-alkyne reactionproceeds slowly, usually requires high temperatures or pressures, andyields a mixture of 1,4- and 1,5-triazoles, all of which render thisreaction generally unsuitable for most applications involvingbiomolecules. Transition metals, in particular copper catalysts, havebeen found to accelerate the azide-alkyne reaction through a differentmechanism and allows for the reaction to proceed under aqueousconditions and at room temperature. Although, non-transition metalcatalyzed variants of the azide-alkyne reaction, which rely on ringstrain rather than transition metal catalysts, have been developed,these methods often require multistep and lengthy syntheses to preparethe necessary reactants. As such, transition metal-catalyzedazide-alkyne cycloaddition, in particular the copper-catalyzedazide-alkyne cycloaddition (CuAAC), has seen widespread use.

Previously, Hong et. al. described a modified procedure for ahomogeneous copper-catalyzed azide-alkyne cycloaddition that provided ageneral straightforward approach for bioconjugation (See Hong et al.,Angew. Chem. Int. Ed. 2009, 48, 9879-9883 and Hong et al., ChemBioChem,2008, 9, 1481-1486). The new protocol provided some importantimprovements—most notably, being able to run the reaction in air insteadof anaerobically; however, a key drawback remaining is the removal ofadded reagents, such as ascorbic acid, aminoguanidine, and thecopper-ligand catalyst complex.

The use of a heterogeneous catalyst for azide-alkyne cycloaddition is anattractive alternative. A heterogeneous catalyst allows for easy productseparation, minimal waste generation during catalyst separation,catalyst recyclability, and facile catalyst removal. Furthermore, aheterogeneous catalyst is especially advantageous for use in biologicalapplications since one of the advantages of a heterogeneous system isfacile removal of the catalyst, which is important especially given thecytotoxic properties of certain transition metals, especially copper.Despite these advantages, heterogeneous metal catalysts that have beenemployed for azide-alkyne cycloaddition still suffer from significantdisadvantages. Some of them include multi-step catalyst preparation, lowloading, long reaction times, harsh reaction conditions, tedious work upof the catalyst and products. All of which limit the utility of aheterogeneous metal catalyst in bioconjugation and for use withbiomolecules.

Described herein are methods for using a heterogeneous metal catalyst,such as copper, for the azide-alkyne cycloaddition. Such methods areused to both synthesize and purify cycloaddition compounds (e.g.,triazoles), including biomolecules, in essentially one step with the useof a chromatography column that contains a matrix comprising aheterogeneous metal catalyst. In the methods described herein, asolution containing the alkyne component and azide component is passedthrough a column containing a matrix comprising the heterogeneous metalcatalyst, wherein upon passing the solution through the column by eithergravity or centrifugation, the alkyne component and azide componentreact upon contact with the heterogeneous metal catalyst in the matrixto form a triazole product. In some embodiments, the method furthercomprises passing the triazole product through an additional matrix. Insome embodiments, the additional matrix is a resin that allows forpurification of the triazole product. In some embodiments, the methodfurther comprises passing the triazole product through an additionalmatrix that is a resin that allows for the purification of the triazoleproduct. In some embodiments, the column is suitable for use in gravitycolumn chromatography or in centrifugal chromatography.

In some of the methods provided herein, the heterogeneous metal catalystis a metal catalyst that catalyzes the azide-alkyne cycloaddition.Examples of such heterogeneous metal catalysts include but are notlimited to copper, ruthenium, silver and zinc. In some of the methodsprovided herein, the heterogeneous metal catalyst comprises a coppercatalyst. In some of the methods provided herein, the heterogeneouscopper catalyst comprises a copper (I) catalyst. In some of the methodsprovided herein, the heterogeneous copper catalyst comprises a copper(II) precatalyst and a reducing agent, wherein the copper (I) catalystis generated from the reduction of the copper (II) precatalyst. In someof the methods provided herein, the reducing agent is zinc amalgam.

One of the advantages of the methods disclosed herein is that thesemethods are useful to chemically modify a wide range of cycloadditioncompounds, including small molecules and biomolecules. Furthermore, insome embodiments, these methods are performed under aqueous conditionsand at room temperature. The chemical modification of biomolecules, suchas proteins, nucleic acids, and carbohydrates, is particularlyadvantageous, and the methods disclosed herein allow for facilebioconjugation through a one-step synthesis and purification process.The use of the disclosed matrix comprising the heterogeneous metalcatalyst is particularly well suited for bioconjugation as it offersfacile product separation and catalyst removal. Also in someembodiments, the use of the disclosed matrix comprising theheterogeneous metal catalyst for bioconjugation also allows for thepurification from cells and cell lysates, wherein the alkyne componentand/or azide component contain cells and/or cell lysates. In someembodiments, the matrix disclosed herein does not allow for the bulk ofthe cells and/or cell lysates to pass through, thereby affording aproduct that is free or substantially free of impurities derived fromcells and/or cell lysates. Also disclosed herein are methods forpreparing glycosylated DNA and fluorescent labeled peptides.Furthermore, compositions and kits related to the methods disclosedherein are also described.

DEFINITIONS

As used herein “alkyne” or “alkynyl” refers to a compound containing acarbon-carbon triple bond functional group. In some embodiments, alkyneor alkynyl refers to a compound containing a mono-substituted orterminal alkyne. In some embodiments, alkyne or alkynyl refers to acompound containing a di-substituted or internal alkyne.

As used herein “azide” or “azido” refers to a compound containing theanion N₃ ⁻, wherein N is nitrogen.

As used herein “column” refers to a tube that is used for the separationof compounds (e.g., cycloaddition compounds) by passing the compoundsthrough a matrix packed within the column in the presence of a suitablesolvent or buffer solution under gravity or centrifugal force. Suchcolumns are further equipped with a matrix barrier, which is at thebottom of the tube and is used to contain the matrix packed within thecolumn. In some embodiments, the matrix barrier is a frit with a nominalpore size. In some embodiments, the matrix barrier is a membrane filter.In some embodiments, the matrix barrier is a cotton or glass wool plug.As used herein, columns are synonymous with chromatography columns andencompass any type of means to pass any suitable solvent, solution, orbuffer solution through the column, such as gravity or centrifugalforce.

As used herein “centrifuge column” refers to a tube that is used for theseparation of compounds (e.g., cycloaddition compounds), which includebiomolecules, by passing the compounds through a matrix packed withinthe column in the presence of a suitable solvent or buffer undercentrifugal force. Such centrifuge columns are further equipped with amatrix barrier, which is at the bottom of the tube and is used tocontain the matrix packed within the column. In some embodiments, thematrix barrier is a frit with a nominal pore size. In some embodiments,the matrix barrier is a membrane filter. As used herein, centrifugecolumns are synonymous with spin columns and encompass any sample volumeand type capable of being separated by centrifugal force.

As used herein “gravity column” refers to a tube that is used for theseparation of compounds (e.g., cycloaddition compounds), which includebiomolecules, by passing the compounds through a matrix packed withinthe column in the presence of a suitable solvent or buffer undergravity. In some embodiments, the flow of the solvent or buffer isfacilitated with the use of positive pressure from air or compressedgas, e.g., flash chromatography. Such gravity columns are furtherequipped with a matrix barrier, which is at the bottom of the tube andis used to contain the matrix packed within the column. In someembodiments, the matrix barrier is a frit with a nominal pore size. Insome embodiments, the matrix barrier is a cotton or glass wool plug. Asused herein, gravity columns encompass any sample volume and typecapable of being separated by gravity force and optionally, positivepressure from air or compressed gas.

As used herein, the Huisgen 1,3 dipolar cycloaddition of azides andalkynes refers to the reaction between an azide or a terminal orinternal alkyne to yield a 1,2,3 triazole. In some embodiments, thereaction yields 1,4-disubstituted [1,2,3]-triazoles, 1,5-disubstituted[1,2,3]-triazoles, or a mixture thereof.

As used herein “copper-catalyzed azide-alkyne cycloaddition” refers tothe Huisgen 1,3-dipolar cycloaddition of azides and alkynes catalyzed bycopper (I). In some embodiments, the copper-catalyzed azide-alkynecycloaddition leads to the formation of 1,4-disubstituted[1,2,3]-triazoles. In some embodiments, the copper-catalyzedazide-alkyne cycloaddition leads to the formation of 1,5-disubstituted[1,2,3]-triazoles. Synonyms include, but are not limited to,copper-catalyzed azide-alkyne coupling, copper-catalyzed azide-alkyneclick reaction, copper-catalyzed click chemistry, and copper-catalyzedazide-alkyne Huisgen [3+2] cycloaddition.

As used herein “metal-catalyzed azide-alkyne cycloaddition” refers tothe Huisgen 1,3-dipolar cycloaddition of azides and alkynes catalyzed bya transition metal capable of catalyzing the azide-alkyne cycloaddition.Examples of such metals include but are not limited to copper, silver,ruthenium, and zinc. In some embodiments, the metal-catalyzedazide-alkyne cycloaddition leads to the formation of 1,4-disubstituted[1,2,3]-triazoles. In some embodiments, the metal-catalyzed azide-alkynecycloaddition leads to the formation of 1,5-disubstituted[1,2,3]-triazoles. Synonyms include, but are not limited to,metal-catalyzed azide-alkyne coupling, metal-catalyzed azide-alkyneclick reaction, metal-catalyzed click chemistry, and metal-catalyzedazide-alkyne Huisgen [3+2] cycloaddition. In some embodiments, the“metal-catalyzed azide-alkyne cycloaddition” is synonymous withheterogeneous metal-catalyzed azide-alkyne cycloaddition orheterogeneous transition metal-catalyzed azide-alkyne cycloaddition.

As used herein “copper (0) precatalyst” is meant to encompass anysuitable copper (0) source that is capable of being oxidized by anoxidizing agent to form a catalytically active copper (I) species. Thiscatalytically active copper (I) species facilitates the copper-catalyzedazide-alkyne cycloaddition reaction.

As used herein “copper (II) precatalyst” is meant to encompass anysuitable copper (II) salt or copper (II) metal-ligand complex that iscapable of being reduced by a reducing agent to form a catalyticallyactive copper (I) species. This catalytically active copper (I) speciesfacilitates the copper-catalyzed azide-alkyne cycloaddition reaction.

As used herein “copper (I) catalyst” is meant to encompass any suitablecopper (I) salt or copper (I) metal-ligand complex that is capable ofcatalyzing the azide-alkyne cycloaddition reaction.

As used herein, an “azide component” is meant to encompass any compound,including any small molecule and biomolecule, that contains an azidecomponent that is able to react with an alkyne component to form a[1,2,3]-triazole. In some embodiments, the azide component is generatedin situ with a suitable azide reagent, such as sodium azide.

As used herein, an “alkyne component” is meant to encompass anycompound, including any small molecule or biomolecule, that contains analkyne component that is able to react with an azide component to form a1,2,3-triazole.

As used herein, an “ion-exchange resin” is meant to encompass aninsoluble matrix or support structure that is capable of trapping ofions with concomitant releasing of other ions. The counterions withinthe resin are mobile and are exchangeable with other counterions whileions of the same charge type are not mobile and remain bound to theresin. Ion-exchange resins are optionally classified based on the chargeof the exchangeable ions. For example, cation-exchange resins are resinsthat have positively charged mobile ions available for exchange.Anion-exchange resins are resins that have negatively charged mobileions available for exchange. Additionally, in some embodiments,ion-exchange resins are a combination of cation-exchange andanion-exchange resins. These resins are optionally further classified bythe ionic strength of the bound ion, such as strong or weak exchangeresins.

As used herein, a “size-exclusion resin” is meant to encompass aninsoluble matrix or support structure that capable or separatingmolecules based on molecular size. The term “gel-filtration resin” issynonymous with size-exclusion resin.

As used herein, a “cell lysate derivatized product” refers to a compoundthat is prepared through the metal catalyzed azide-alkyne cycloadditiondisclosed herein, wherein the alkyne component and/or azide componentcontains cells and/or cell lysates. When the solution containing thealkyne component and azide component and cells and/or cell lysates ispassed through any one of the columns described herein, the majority ofthe cells and/or cell lysates remain at the top of the column and do notpass through, thereby providing a cell lysate derivatized product, e.gbioconjugated product, that is free or substantially free of impuritiesderived from the cells and/or cell lysates. In some instances, the celllysate derivatized product contains trace impurities derived from thecells and/or cell lysates and require further purification to removethese impurities. These impurities include but are not limited toproteins and related materials that are derived from cells and/or celllysates that are soluble and are able to pass through any one of thecolumn matrices described herein.

As used herein “free or substantially free” in reference to a particularcompound refers that the amount of said compound is present in about 10ppm or less. In some instances, the said compound is present in about 5ppm or less. In some instances, the compound is present in about 1 ppmor less.

Methods

The methods disclosed herein encompass the synthesis and purification ofcycloaddition compounds, including biomolecules, prepared through theheterogeneous metal-catalyzed azide-alkyne cycloaddition utilizing amatrix comprising a heterogeneous metal catalyst. The matrix iscontained within a column, a tube that contains a matrix barrier at thebottom of the column. The matrix barrier is used to contain the matrixwithin the column. In some instances, the matrix barrier is a frit witha nominal pore size. In other instances, the matrix barrier is amembrane filter. In other instances, the matrix barrier is a cotton orglass wool plug. These methods allow for the addition of the reactivecomponents—the addition of the alkyne containing compound and the azidecontaining compound to the column. Immobilization of the alkyne andazide components on to the matrix is accomplished through passing thesolution containing the alkyne and azide components under gravity orcentrifugal force, wherein upon contact with the heterogeneous metalcatalyst within the matrix, the alkyne and azide react to form thetriazole product. The triazole product is then eluted with anappropriate buffer solution, solvent, or solution. In some embodiments,purification is achieved by incorporating an additional matrix to thecolumn such that once the triazole product has been formed in matrixcomprising that heterogeneous metal catalyst, the triazole product iseluted through the additional matrix comprising a resin suitable forfurther purifying the product, such as a size-exclusion resin.

In some embodiments, the triazole is a cell lysate derivatized product.In some embodiments, the cell lysate derivatized product is free orsubstantially free of impurities derived from cells and/or cell lysates.In some embodiments, the impurities include but are not limited toproteins and related materials derived from cells and/or cell lysatesthat are able to pass through the column matrix. In some embodiments,the cell lysate derivatized product requires secondary purification toremove trace impurities derived from cells and/or cell lysates. In someembodiments, the cell lysate derivatized product is free orsubstantially free of impurities derived from cell lysates. In someembodiments, the impurities include but are not limited to proteins andrelated materials derived from cell lysates that are able to passthrough the column matrix. In some embodiments, the cell lysatederivatized product requires secondary purification to remove traceimpurities derived from cell lysates. In some embodiments, secondarypurification is any one of the methods disclosed herein for additionalpurification.

The heterogeneous metal catalyst for these methods described hereincomprises a heterogeneous metal catalyst that is suitable for catalyzingthe azide-alkyne cycloaddition. Examples of such heterogeneous metalscatalysts include, but are not limited to, copper, ruthenium, silver andzinc.

Provided herein is a method for preparing a cycloaddition compound(e.g., a triazole from the azide-alkyne cycloaddition) from a reactioncatalyzed by a heterogeneous copper catalyst, comprising mixing analkyne component and an azide component in an appropriate solvent toform a solution; transferring the solution containing the alkynecomponent and the azide component to a column with a matrix barrier atthe bottom of the column; wherein the column comprises a matrix of acopper (I) catalyst; and passing the solution containing the alkynecomponent and the azide component through the column; wherein uponcontact with the copper (I) catalyst within the matrix, the alkynecomponent and the azide component react to form the cycloadditioncompound.

Also provided herein is a method for preparing a cycloaddition compound(e.g., a triazole from the azide-alkyne cycloaddition) from a reactioncatalyzed by a heterogeneous copper catalyst, comprising mixing analkyne component and an azide component in an appropriate solvent toform a solution; transferring the solution containing the alkynecomponent and the azide component to a column with a matrix barrier atthe bottom of the column; wherein the column comprises a matrix of acopper (II) precatalyst and a reducing agent; wherein a copper (I)catalyst is generated from the reduction of the copper (II) precatalystwith the reducing agent; and passing the solution containing the alkynecomponent and the azide component through the column; wherein uponcontact with the copper (I) catalyst within the matrix, the alkynecomponent and the azide component react to form the cycloadditioncompound. In some embodiments, the reducing agent is zinc amalgam.

Additionally, provided herein is a method for preparing a cycloadditioncompound (e.g., a triazole from the azide-alkyne cycloaddition) from areaction catalyzed by a heterogeneous copper catalyst, comprising mixingan alkyne component and an azide component in an appropriate solvent toform a solution; transferring the solution containing the alkynecomponent and the azide component to a column with a matrix barrier atthe bottom of the column; wherein the column comprises a matrix of acopper (II) precatalyst and zinc amalgam; wherein a copper (I) catalystis generated from the reduction of the copper (II) precatalyst with zincamalgam; and passing the solution containing the alkyne component andthe azide component through the column; wherein upon contact with thecopper (I) catalyst within the matrix, the alkyne component and theazide component react to form the cycloaddition compound.

In some embodiments, the column is suitable for use in gravity columnchromatography or centrifugal column chromatography. In someembodiments, passing the solution containing the alkyne component andthe azide component through the column is through gravity. In furtherembodiments, passing the solution containing the alkyne component andthe azide component through the column further comprises the use ofpositive pressure from air or a compressed gas. In some embodiments,passing the solution containing the alkyne component and the azidecomponent through the column is through centrifuging the column for asufficient period of time.

In some embodiments, the column further comprises an additional matrix.In some embodiments, the column further comprises an additional matrixsuitable for purification of the cycloaddition compound. In someembodiments, the additional matrix is a resin. In some embodiments, thecolumn further comprises a resin suitable for purification of theproduct. In some embodiments, the resin is size-exclusion resin. In someembodiments, the column further comprises a resin. In some embodiments,the column further comprises a resin suitable for the purification ofthe cycloaddition compound. In some embodiments, the resin issize-exclusion resin.

In some embodiments, the column further comprises an additional matrixthat is a resin suitable for the purification of the cycloadditioncompound. In some embodiments, the resin is size-exclusion resin.

In some embodiments, the cycloaddition compound is eluted with anappropriate solvent. In some embodiments, the cycloaddition compound iseluted with an appropriate solvent by passing the solvent through thecolumn through gravity. In some embodiments, wherein the cycloadditioncompound is eluted with an appropriate solvent by passing the solventthrough the column through gravity, positive pressure from air orcompressed gas is also used to facilitate solvent flow. In someembodiments, the cycloaddition compound is eluted with an appropriatesolvent by passing the solvent through the column throughcentrifugation. In some embodiments, the cycloaddition compound iseluted with an appropriate buffer solution. In some embodiments, thecycloaddition compound is eluted with an appropriate buffer solution bypassing the buffer solution through the column through gravity. In someembodiments, wherein the cycloaddition compound is eluted with anappropriate buffer solution by passing the buffer solution through thecolumn through gravity, positive pressure from air or compressed gas isalso used to facilitate solvent flow. In some embodiments, thecycloaddition compound is eluted with an appropriate buffer solution bypassing the buffer solution through the column through centrifugation.

Provided herein is a method for preparing a cycloaddition compound froma reaction catalyzed by a heterogeneous copper catalyst, comprisingmixing an alkyne component and an azide component in an appropriatesolvent to form a solution; transferring the solution containing thealkyne component and the azide component to a gravity column with amatrix barrier at the bottom of the gravity column; wherein the gravitycolumn comprises a matrix of a copper (I) catalyst; and passing thesolution containing the alkyne component and the azide component throughthe gravity column; wherein upon contact with the copper (I) catalystwithin the matrix, the alkyne component and the azide component react toform the cycloaddition compound.

Also, provided herein is a method for preparing a cycloaddition compound(e.g., a triazole from the azide-alkyne cycloaddition) from a reactioncatalyzed by a heterogeneous copper catalyst, comprising mixing analkyne component and an azide component in an appropriate solvent toform a solution; transferring the solution containing the alkynecomponent and the azide component to a gravity column with a matrixbarrier at the bottom of the gravity column; wherein the gravity columncomprises a matrix of a copper (II) precatalyst and a reducing agent;wherein a copper (I) catalyst is generated from the reduction of thecopper (II) precatalyst with the reducing agent; and passing thesolution containing the alkyne component and the azide component throughthe gravity column; wherein upon contact with the copper (I) catalystwithin the matrix, the alkyne component and the azide component react toform the cycloaddition compound.

Also, provided herein is a method for preparing a cycloaddition compound(e.g., a triazole from the azide-alkyne cycloaddition) from a reactioncatalyzed by a heterogeneous copper catalyst, comprising mixing analkyne component and an azide component in an appropriate solvent toform a solution; transferring the solution containing the alkynecomponent and the azide component to a gravity column with a matrixbarrier at the bottom of the gravity column; wherein the gravity columncomprises a matrix of a copper (II) precatalyst and zinc amalgam;wherein a copper (I) catalyst is generated from the reduction of thecopper (II) precatalyst with zinc amalgam; and passing the solutioncontaining the alkyne component and the azide component through thegravity column; wherein upon contact with the copper (I) catalyst withinthe matrix, the alkyne component and the azide component react to formthe cycloaddition compound.

In some embodiments, the cycloaddition compound is eluted with anappropriate solvent with the use of positive pressure from air or acompressed gas to facilitate solvent flow.

In some embodiments, the method further comprises passing the solutioncontaining the cycloaddition compound through an additional matrix. Insome embodiments, the additional matrix is a resin suitable for thepurification of the cycloaddition compound. In some embodiments, themethod further comprises pass the solution containing the compoundthrough an additional matrix that is suitable for the purification ofthe cycloaddition compound. In some embodiments, the additional matrixis a size-exclusion matrix. In some embodiments, the solution containingthe alkyne component and azide component is aqueous.

Provided herein is a method for preparing a cycloaddition compound(e.g., a triazole from the azide-alkyne cycloaddition) from a reactioncatalyzed by a heterogeneous copper catalyst, comprising mixing analkyne component and an azide component in an appropriate solvent toform a solution; transferring the solution containing the alkynecomponent and the azide component to a centrifuge column with a matrixbarrier at the bottom of the centrifuge column; wherein the centrifugecolumn comprises a matrix of a copper (I) catalyst; and passing thesolution containing the alkyne component and the azide component throughthe centrifuge column by centrifuging the centrifuge column for asufficient period of time; wherein upon contact with the copper (I)catalyst within the matrix, the alkyne component and the azide componentreact to form the cycloaddition compound.

Also provided herein is a method for preparing a cycloaddition compound(e.g., a triazole from the azide-alkyne cycloaddition) from a reactioncatalyzed by a heterogeneous copper catalyst, comprising mixing analkyne component and an azide component in an appropriate solvent toform a solution; transferring the solution containing the alkynecomponent and the azide component to a centrifuge column with a matrixbarrier at the bottom of the centrifuge column; wherein the centrifugecolumn comprises a matrix of a copper (II) precatalyst and a reducingagent; wherein a copper (I) catalyst is generated from the reduction ofthe copper (II) precatalyst with the reducing agent; and passing thesolution containing the alkyne component and the azide component throughcentrifuge column by centrifuging the centrifuge column for a sufficientperiod of time; wherein upon contact with the copper (I) catalyst withinthe matrix, the alkyne component and the azide component react to formthe cycloaddition compound. In some embodiments, the reducing agent iszinc amalgam.

Additionally, provided herein is a method for preparing a cycloadditioncompound (e.g., a triazole from the azide-alkyne cycloaddition) from areaction catalyzed by a heterogeneous copper catalyst, comprising mixingan alkyne component and an azide component in an appropriate solvent toform a solution; transferring the solution containing the alkynecomponent and the azide component to a centrifuge column with a matrixbarrier at the bottom of the centrifuge column; wherein the centrifugecolumn comprises a matrix of a copper (II) precatalyst and zinc amalgam;wherein a copper (I) catalyst is generated from the reduction of thecopper (II) precatalyst with zinc amalgam; and passing the solutioncontaining the alkyne component and the azide component through thecentrifuge column by centrifuging the centrifuge column for a sufficientperiod of time; wherein upon contact with the copper (I) catalyst withinthe matrix, the alkyne component and the azide component react to formthe cycloaddition compound.

In some embodiments, the cycloaddition compound is eluted with anappropriate buffer solution followed by centrifuging the centrifugecolumn for a sufficient period of time.

In some embodiments, the method further comprises passing the solutioncontaining the cycloaddition compound through an additional matrix. Insome embodiments, the additional matrix is a resin suitable for thepurification of the cycloaddition compound. In some embodiments, themethod further comprises passing the solution containing thecycloaddition compound through an additional matrix that is a resinsuitable for the purification of the cycloaddition compound. In someembodiments, the additional matrix is a size-exclusion matrix. In someembodiments, the solution containing the alkyne component and azidecomponent is aqueous.

In some embodiments, the cycloaddition compound is a cell lysatederivatized product. In some embodiments, the cell lysate derivatizedproduct is free or substantially free of impurities derived from cellsand/or cell lysates. In some embodiments, the impurities include but arenot limited to proteins and related materials derived from cells and/orcell lysates that are able to pass through the column matrix. In someembodiments, the cell lysate derivatized product requires secondarypurification to remove trace impurities derived from cells and/or celllysates. In some embodiments, the cell lysate derivatized product isfree or substantially free of impurities derived from cell lysates. Insome embodiments, the impurities include but are not limited to proteinsand related materials derived from cell lysates that are able to passthrough the column matrix. In some embodiments, the cell lysatederivatized product requires secondary purification to remove traceimpurities derived from cell lysates. In some embodiments, secondarypurification is any one of the methods disclosed herein for additionalpurification.

Similarly, the methods disclosed herein allow for the synthesis andpurification of cycloaddition compounds, including biomolecules,prepared through the heterogeneous metal-catalyzed azide-alkynecycloaddition utilizing a matrix comprising a heterogeneous metalcatalyst, wherein the alkyne component and/or azide component containcells and/or cell lysates. In some instances, the alkyne and/or alkynecomponents contain cell lysates. In some instances, passing the solutioncontaining the alkyne component and azide component through the columnallows for the majority of the cells and/or cell lysates to remain onthe top of the column and to not pass through, thereby affording aresultant product that is relatively free of impurities derived fromcells and/or cell lysates. In some instances, further purification maybe required for the resultant product to remove trace impurities.

Provided herein is a method for preparing a cell lysate derivatizedproduct from a reaction catalyzed by a heterogeneous copper catalyst,comprising mixing an alkyne component and an azide component in anappropriate solvent to form a solution; transferring the solutioncontaining the alkyne component and the azide component to a column witha matrix barrier at the bottom of the column; wherein the columncomprises a matrix of a copper (I) catalyst; and passing the solutioncontaining the alkyne component and the azide component through thecolumn; wherein upon contact with the copper (I) catalyst within thematrix, the alkyne component and the azide component react to form thecell lysate derivatized product.

Also provided herein is a method for preparing a cell lysate derivatizedproduct from a reaction catalyzed by a heterogeneous copper catalyst,comprising mixing an alkyne component and an azide component in anappropriate solvent to form a solution; transferring the solutioncontaining the alkyne component and the azide component to a column witha matrix barrier at the bottom of the column; wherein the columncomprises a matrix of a copper (II) precatalyst and a reducing agent;wherein a copper (I) catalyst is generated from the reduction of thecopper (II) precatalyst with the reducing agent; and passing thesolution containing the alkyne component and the azide component throughthe column; wherein upon contact with the copper (I) catalyst within thematrix, the alkyne component and the azide component react to form thecell lysate derivatized product. In some embodiments, the reducing agentis zinc amalgam.

Additionally, provided herein is a method for preparing a cell lysatederivatized product from a reaction catalyzed by a heterogeneous coppercatalyst, comprising mixing an alkyne component and an azide componentin an appropriate solvent to form a solution; transferring the solutioncontaining the alkyne component and the azide component to a column witha matrix barrier at the bottom of the column; wherein the columncomprises a matrix of a copper (II) precatalyst and zinc amalgam;wherein a copper (I) catalyst is generated from the reduction of thecopper (II) precatalyst with zinc amalgam; and passing the solutioncontaining the alkyne component and the azide component through thecolumn; wherein upon contact with the copper (I) catalyst within thematrix, the alkyne component and the azide component react to form thecell lysate derivatized product.

In some embodiments, the column is suitable for use in gravity columnchromatography or centrifugal column chromatography. In someembodiments, passing the solution containing the alkyne component andthe azide component through the column is through gravity. In furtherembodiments, passing the solution containing the alkyne component andthe azide component through the column further comprises the use ofpositive pressure from air or a compressed gas. In some embodiments,passing the solution containing the alkyne component and the azidecomponent through the column is through centrifuging the column for asufficient period of time.

In some embodiments, the column further comprises an additional matrix.In some embodiments, the column further comprises an additional matrixsuitable for purification of the cell lysate derivatized product. Insome embodiments, the additional matrix is a resin. In some embodiments,the column further comprises a resin suitable for purification of theproduct. In some embodiments, the resin is size-exclusion resin. In someembodiments, the column further comprises a resin. In some embodiments,the column further comprises a resin suitable for the purification ofthe cell lysate derivatized product. In some embodiments, the resin issize-exclusion resin. In some embodiments, the column further comprisesan additional matrix that is a resin that is suitable for thepurification of the cell lysate derivatized product. In someembodiments, the additional matrix is a size-exclusion resin.

In some embodiments, the cell lysate derivatized product is eluted withan appropriate solvent. In some embodiments, the cell lysate derivatizedproduct is eluted with an appropriate solvent by passing the solventthrough the column through gravity. In some embodiments, wherein thecell lysate derivatized product is eluted with an appropriate solvent bypassing the solvent through the column through gravity, positivepressure from air or compressed gas is also used to facilitate solventflow. In some embodiments, the cell lysate derivatized product is elutedwith an appropriate solvent by passing the solvent through the columnthrough centrifugation. In some embodiments, the cell lysate derivatizedproduct is eluted with an appropriate buffer solution. In someembodiments, the cell lysate derivatized product is eluted with anappropriate buffer solution by passing the buffer solution through thecolumn through gravity. In some embodiments, wherein the cell lysatederivatized product is eluted with an appropriate buffer solution bypassing the buffer solution through the column through gravity, positivepressure from air or compressed gas is also used to facilitate solventflow. In some embodiments, the cell lysate derivatized product is elutedwith an appropriate buffer solution by passing the buffer solutionthrough the column through centrifugation.

Provided herein is a method for preparing a cell lysate derivatizedproduct from a reaction catalyzed by a heterogeneous copper catalyst,comprising mixing an alkyne component and an azide component in anappropriate solvent to form a solution; transferring the solutioncontaining the alkyne component and the azide component to a gravitycolumn with a matrix barrier at the bottom of the gravity column;wherein the gravity column comprises a matrix of a copper (I) catalyst;and passing the solution containing the alkyne component and the azidecomponent through the gravity column; wherein upon contact with thecopper (I) catalyst within the matrix, the alkyne component and theazide component react to form the cell lysate derivatized product.

Also, provided herein is a method for preparing a cell lysatederivatized product from a reaction catalyzed by a heterogeneous coppercatalyst, comprising mixing an alkyne component and an azide componentin an appropriate solvent to form a solution; transferring the solutioncontaining the alkyne component and the azide component to a gravitycolumn with a matrix barrier at the bottom of the gravity column;wherein the gravity column comprises a matrix of a copper (II)precatalyst and a reducing agent; wherein a copper (I) catalyst isgenerated from the reduction of the copper (II) precatalyst with thereducing agent; and passing the solution containing the alkyne componentand the azide component through the gravity column; wherein upon contactwith the copper (I) catalyst within the matrix, the alkyne component andthe azide component react to form the cell lysate derivatized product.

Also, provided herein is a method for preparing a cell lysatederivatized product from a reaction catalyzed by a heterogeneous coppercatalyst, comprising mixing an alkyne component and an azide componentin an appropriate solvent to form a solution; transferring the solutioncontaining the alkyne component and the azide component to a gravitycolumn with a matrix barrier at the bottom of the gravity column;wherein the gravity column comprises a matrix of a copper (II)precatalyst and zinc amalgam; wherein a copper (I) catalyst is generatedfrom the reduction of the copper (II) precatalyst with zinc amalgam; andpassing the solution containing the alkyne component and the azidecomponent through the gravity column; wherein upon contact with thecopper (I) catalyst within the matrix, the alkyne component and theazide component react to form a cell lysate derivatized product.

In some embodiments, the cell lysate derivatized product is eluted withan appropriate solvent with the use of positive pressure from air or acompressed gas to facilitate solvent flow.

In some embodiments, the method further comprises passing the solutioncontaining the cell lysate derivatized product through an additionalmatrix. In some embodiments, the additional matrix is a resin suitablefor the purification of the cell lysate derivatized product. In someembodiments, the method further comprises passing the solutioncontaining the cell lysate derivatized product through an additionalmatrix that is a resin suitable for the purification of the cell lysatederivatized product. In some embodiments, the additional matrix is asize-exclusion matrix. In some embodiments, the solution containing thealkyne component and azide component is aqueous.

Provided herein is a method for preparing a cell lysate derivatizedproduct from a reaction catalyzed by a heterogeneous copper catalyst,comprising mixing an alkyne component and an azide component in anappropriate solvent to form a solution; transferring the solutioncontaining the alkyne component and the azide component to a centrifugecolumn with a matrix barrier at the bottom of the centrifuge column;wherein the centrifuge column comprises a matrix of a copper (I)catalyst; and passing the solution containing the alkyne component andthe azide component through the centrifuge column by centrifuging thecentrifuge column for a sufficient period of time; wherein upon contactwith the copper (I) catalyst within the matrix, the alkyne component andthe azide component react to form the cell lysate derivatized product.

Also provided herein is a method for preparing a cell lysate derivatizedproduct from a reaction catalyzed by a heterogeneous copper catalyst,comprising mixing an alkyne component and an azide component in anappropriate solvent to form a solution; transferring the solutioncontaining the alkyne component and the azide component to a centrifugecolumn with a matrix barrier at the bottom of the centrifuge column;wherein the centrifuge column comprises a matrix of a copper (II)precatalyst and a reducing agent; wherein a copper (I) catalyst isgenerated from the reduction of the copper (II) precatalyst with thereducing agent; and passing the solution containing the alkyne componentand the azide component through centrifuge column by centrifuging thecentrifuge column for a sufficient period of time; wherein upon contactwith the copper (I) catalyst within the matrix, the alkyne component andthe azide component react to form the cell lysate derivatized product.In some embodiments, the reducing agent is zinc amalgam.

Additionally, provided herein is a method for preparing a cell lysatederivatized product from a reaction catalyzed by a heterogeneous coppercatalyst, comprising mixing an alkyne component and an azide componentin an appropriate solvent to form a solution; transferring the solutioncontaining the alkyne component and the azide component to a centrifugecolumn with a matrix barrier at the bottom of the centrifuge column;wherein the centrifuge column comprises a matrix of a copper (II)precatalyst and zinc amalgam; wherein a copper (I) catalyst is generatedfrom the reduction of the copper (II) precatalyst with zinc amalgam; andpassing the solution containing the alkyne component and the azidecomponent through the centrifuge column by centrifuging the centrifugecolumn for a sufficient period of time; wherein upon contact with thecopper (I) catalyst within the matrix, the alkyne component and theazide component react to form the cell lysate derivatized product.

In some embodiments, the cell lysate derivatized product is eluted withan appropriate buffer solution followed by centrifuging the centrifugecolumn for a sufficient period of time.

In some embodiments, the method further comprises passing the solutioncontaining the cell lysate derivatized product through an additionalmatrix. In some embodiments, the additional matrix is a resin suitablefor the purification of the cell lysate derivatized product. In someembodiments, the method further comprises passing the solutioncontaining the cell lysate derivatized product through an additionalmatrix that is a resin suitable for the purification of the cell lysatederivatized product. In some embodiments, the additional matrix is asize-exclusion matrix. In some embodiments, the solution containing thealkyne component and azide component is aqueous.

In some embodiments, the cell lysate derivatized product is free orsubstantially free of impurities derived from cells and/or cell lysates.In some embodiments, the impurities include but are not limited toproteins and related materials derived from cells and/or cell lysatesthat are able to pass through the column matrix. In some embodiments,the cell lysate derivatized product requires secondary purification toremove trace impurities derived from cells and/or cell lysates. In someembodiments, the cell lysate derivatized product is free orsubstantially free of impurities derived from cell lysates. In someembodiments, the impurities include but are not limited to proteins andrelated materials derived from cell lysates that are able to passthrough the column matrix. In some embodiments, the cell lysatederivatized product requires secondary purification to remove traceimpurities derived from cell lysates. In some embodiments, secondarypurification is any one of the methods disclosed herein for additionalpurification.

Azide Component

Azide components contemplated for use include any compound, includingany biomolecule that contains an azide moiety. Suitable azide componentsalso include compounds that have a functional group, such as a benzylhalide or α-halo ketone that is capable with reacting with an azidereagent, such as sodium azide, to form an azide in situ. The alkynecomponent is optionally any organic compound, small molecule, nucleicacid, amino acid, carbohydrate, fluorophore, or antibody that containsan alkyne.

In some embodiments, the azide component is an organic compound. In someembodiments, the azide component comprises a small molecule, a nucleicacid, an amino acid, a carbohydrate, a fluorophore, or an antibody. Insome embodiments, the azide component comprises a small molecule, aprotein, a peptide, an amino acid, an oligonucleotide, a nucleotide, anucleoside, a carbohydrate, or a fluorophore. In some embodiments, theazide component comprises a small molecule. In some embodiments, theazide component comprises an antibody. In some embodiments, the azidecomponent comprises a protein. In some embodiments, the azide componentcomprises a peptide. In some embodiments, the azide component comprisesan amino acid. In some embodiments, the azide component comprises anoligonucleotide. In some embodiments, the azide component comprises anucleotide. In some embodiments, the azide component comprises anucleoside. In some embodiments, the azide component comprises acarbohydrate. In some embodiments, the azide component comprises afluorophore.

In some embodiments, the azide component contains cells and/or celllysates. In some embodiments, the azide component contains cells. Insome embodiments, the azide component contains cell lysates.

Alkyne Component

Alkyne components contemplated for use include any compound orbiomolecule that contains an alkyne moiety. The alkyne component isoptionally any organic compound, small molecule, nucleic acid, aminoacid, carbohydrate, fluorophore, or antibody that contains an alkyne.

In some embodiments, the alkyne component is an organic compound. Insome embodiments, the alkyne component comprises a small molecule, anucleic acid, an amino acid, a carbohydrate, a fluorophore, or anantibody. In some embodiments, the alkyne component comprises a smallmolecule, a protein, a peptide, an amino acid, an oligonucleotide, anucleotide, a nucleoside, a carbohydrate, or a fluorophore. In someembodiments, the alkyne component comprises a small molecule. In someembodiments, the alkyne component comprises an antibody. In someembodiments, the alkyne component comprises a protein. In someembodiments, the alkyne component comprises a peptide. In someembodiments, the alkyne component comprises an amino acid. In someembodiments, the alkyne component comprises an oligonucleotide. In someembodiments, the alkyne component comprises a nucleotide. In someembodiments, the alkyne component comprises a nucleoside. In someembodiments, the alkyne component comprises a carbohydrate. In someembodiments, the alkyne component comprises a fluorophore.

In some embodiments, the alkyne component comprises an oligonucleotideand the azide component comprises a carbohydrate. In some embodiments,the alkyne component comprises a carbohydrate and the azide componentcomprises an oligonucleotide. In some embodiments, alkyne componentcomprises a peptide and the azide component comprises a fluorophore.

In some embodiments, the alkyne component contains cells and/or celllysates. In some embodiments, the alkyne component contains cells. Insome embodiments, the alkyne component contains cell lysates.

Azide-Alkyne Cycloaddition

The heterogeneous metal catalyzed cycloaddition or [3+2] of an alkynecomponent with the azide component forms a [1,2,3]-triazole (alsoreferenced herein as a cycloaddition compound). In some embodiments, the[1,2,3] triazole is a 1,4 disubstituted [1,2,3]-triazole. In someembodiments, the [1,2,3]-triazole is a 1,5-disubstituted[1,2,3]-triazole. In embodiments, the product is a mixture of 1,4disubstituted [1,2,3]-triazole and 1,5-disubstituted [1,2,3]-triazoles.

The copper-catalyzed cycloaddition or [3+2] of an alkyne component withthe azide component forms a [1,2,3]-triazole. In some embodiments, the[1,2,3] triazole is a 1,4 disubstituted [1,2,3]-triazole. In someembodiments, the [1,2,3]-triazole is a 1,5-disubstituted[1,2,3]-triazole.

In some embodiments, the triazole is eluted by loading the column withan appropriate solvent or buffer solution and the solvent or buffersolution is passed through the column by gravity or centrifugation. Insome embodiments, the triazole is eluted by loading the column with anappropriate solvent or buffer solution and the solvent or buffersolution is passed through the column by gravity. In some embodiments,wherein the triazole is eluted by loading the column with an appropriatesolvent or buffer solution and the solvent or buffer solution is passedthrough the column by gravity, positive pressure from air or acompressed gas is also used to facilitate solvent or buffer solutionflow. In some embodiments, the triazole is eluted by loading the columnwith an appropriate solvent or buffer solution and the solvent or buffersolution is passed through the column by centrifugation.

In some embodiments, the triazole is eluted by loading the centrifugecolumn with an appropriate buffer solution followed by centrifugation.In some embodiments, the trizole is eluted with an appropriate buffersolution followed by centrifuging the centrifuge column for a sufficientperiod of time.

In some embodiments, the triazole is a cell lysate derivatized product.In some embodiments, the cell lysate derivatized product is free orsubstantially free of impurities derived from cells and/or cell lysates.In some embodiments, the impurities include but are not limited toproteins and related materials derived from cells and/or cell lysatesthat are able to pass through the column matrix. In some embodiments,the cell lysate derivatized product requires secondary purification toremove trace impurities derived from cells and/or cell lysates. In someembodiments, the cell lysate derivatized product is free orsubstantially free of impurities derived from cell lysates. In someembodiments, the impurities include but are not limited to proteins andrelated materials derived from cell lysates that are able to passthrough the column matrix. In some embodiments, the cell lysatederivatized product requires secondary purification to remove traceimpurities derived from cell lysates. In some embodiments, secondarypurification is any one of the methods disclosed herein for additionalpurification.

Provided herein is a method for preparing a glycosylated DNA from areaction catalyzed by a heterogeneous copper catalyst, comprising mixingan alkynyl DNA component and an azido carbohydrate component in anappropriate solvent to form a solution; transferring the solutioncontaining the alkynyl DNA component and the azido carbohydratecomponent to a column with a matrix barrier at the bottom of the column;wherein the column comprises a matrix of a copper (II) precatalyst and areducing agent; wherein a copper (I) catalyst is generated from thereduction of the copper (II) precatalyst with the reducing agent; andpassing the solution containing the alkynyl DNA component and the azidocarbohydrate component; wherein upon contact with the copper (I)catalyst within the matrix, the alkynyl DNA component and the azidocarbohydrate component react to form a glycosylated DNA.

Provided herein is a method for preparing a glycosylated DNA from areaction catalyzed by a heterogeneous copper catalyst, comprising mixingan alkynyl DNA component and an azido carbohydrate component in anappropriate solvent to form a solution; transferring the solutioncontaining the alkynyl DNA component and the azido carbohydratecomponent to a centrifuge column with a matrix barrier at the bottom ofthe column; wherein the centrifuge column comprises a matrix of a copper(II) precatalyst and a reducing agent; wherein a copper (I) catalyst isgenerated from the reduction of the copper (II) precatalyst with thereducing agent; and passing the solution containing the alkynyl DNAcomponent and the azido carbohydrate component through the centrifugecolumn by centrifuging the centrifuge column for a sufficient period oftime; wherein upon contact with the copper (I) catalyst within thematrix, the alkynyl DNA component and the azido carbohydrate componentreact to form a glycosylated DNA.

Azido carbohydrates contemplated for use include any carbohydrates thatcontain an azide moiety. In some embodiments, the azido carbohydratecontains cells and/or cell lysates. In some embodiments, the azidocarbohydrate contains cell lysates. Alkynyl DNA contemplated for useinclude any DNA that contain an alkynyl moiety. In some embodiments, thealkynyl DNA contains cells and/or cell lysates. In some embodiments, thealkynyl DNA contains cell lysates.

The copper catalyzed cycloaddition or [3+2] of an alkynyl DNA with theazido carbohydrate forms a glycosylated DNA. In some embodiments, theglycosylated DNA is a [1,2,3]-triazole. In some embodiments, the [1,2,3]triazole is a 1,4 disubstituted [1,2,3]-triazole. In some embodiments,the [1,2,3]-triazole is a 1,5-disubstituted [1,2,3]-triazole.

Once the glycosylated DNA is formed, the glycosylated DNA is eluted byloading the centrifuge column with an appropriate buffer solutionfollowed by centrifugation. In some embodiments, the glycosylated DNA iseluted with an appropriate buffer solution followed by centrifuging thecentrifuge column for a sufficient period of time.

In some embodiments, the glycosylated DNA is a cell lysate derivatizedproduct. In some embodiments, the cell lysate derivatized product isfree or substantially free of impurities derived from cells and/or celllysates. In some embodiments, the impurities include but are not limitedto proteins and related materials derived from cells and/or cell lysatesthat are able to pass through the column matrix. In some embodiments,the cell lysate derivatized product requires secondary purification toremove trace impurities derived from cells and/or cell lysates. In someembodiments, the cell lysate derivatized product is free orsubstantially free of impurities derived from cell lysates. In someembodiments, the impurities include but are not limited to proteins andrelated materials derived from cell lysates that are able to passthrough the column matrix. In some embodiments, the cell lysatederivatized product requires secondary purification to remove traceimpurities derived from cell lysates. In some embodiments, secondarypurification is any one of the methods disclosed herein for additionalpurification.

Provided herein is a method for preparing a fluorescent labeled peptidefrom a reaction catalyzed by a heterogeneous copper catalyst, comprisingmixing an alkynyl peptide component and an azido fluorogenic dyecomponent in an appropriate solvent to form a solution; transferring thesolution containing the alkynyl peptide component and the azidofluorogenic dye component to a column with a matrix barrier at thebottom of the column; wherein the column comprises a matrix of copper(II) precatalyst and a reducing agent; wherein a copper (I) catalyst isgenerated from the reduction of the copper (II) precatalyst with thereducing agent; and passing the solution containing the alkynyl peptidecomponent and the azido fluorogenic dye component through the column;wherein upon contact with the copper (I) catalyst within the matrix, thealkynyl peptide component and the azido fluorogenic component react toform a fluorescent labeled peptide.

Provided herein is a method for preparing a fluorescent labeled peptidefrom a reaction catalyzed by a heterogeneous copper catalyst, comprisingmixing an alkynyl peptide component and an azido fluorogenic dyecomponent in an appropriate solvent to form a solution; transferring thesolution containing the alkynyl peptide component and the azidofluorogenic dye component to a centrifuge column with a matrix barrierat the bottom of the column; wherein the centrifuge column comprises amatrix of copper (II) precatalyst and a reducing agent; wherein a copper(I) catalyst is generated from the reduction of the copper (II)precatalyst with the reducing agent; and passing the solution containingthe alkynyl peptide component and the azido fluorogenic dye componentthrough the centrifuge column by centrifuging the centrifuge column fora sufficient period of time; wherein upon contact with the copper (I)catalyst within the matrix, the alkynyl peptide component and the azidofluorogenic component react to form a fluorescent labeled peptide.

Azido fluorogenic dyes contemplated for use include any fluorogenic dyethat contains an azide moiety. In some embodiments, the azidofluorogenic dye contains cells and/or cell lysates. In some embodiments,the azido fluorogenic dye contains cell lysates. Alkynyl peptidescontemplated for use include any peptide that contains an alkynylmoiety. In some embodiments, the alkynyl peptide contains cells and/orcell lysates. In some embodiments, the alkynyl peptide contains celllysates. The copper catalyzed cycloaddition or [3+2] of an alkynylpeptide with the azido fluorogenic dye forms a fluorescent labeledpeptide. In some embodiments, the fluorescent label peptide is a[1,2,3]-triazole. In some embodiments, the [1,2,3] triazole is a 1,4disubstituted [1,2,3]-triazole. In some embodiments, the[1,2,3]-triazole is a 1,5-disubstituted [1,2,3]-triazole.

Once the fluorescent labeled peptide is formed, the fluorescent labeledpeptide is eluted by loading the centrifuge column with an appropriatebuffer solution followed by centrifugation. In some embodiments, thefluorescent labeled peptide is eluted with an appropriate buffersolution followed by centrifuging the centrifuge column for a sufficientperiod of time.

In some embodiments, the fluorescent labeled peptide is a cell lysatederivatized product. In some embodiments, the cell lysate derivatizedproduct is free or substantially free of impurities derived from cellsand/or cell lysates. In some embodiments, the impurities include but arenot limited to proteins and related materials derived from cells and/orcell lysates that are able to pass through the column matrix. In someembodiments, the cell lysate derivatized product requires secondarypurification to remove trace impurities derived from cells and/or celllysates. In some embodiments, the cell lysate derivatized product isfree or substantially free of impurities derived from cell lysates. Insome embodiments, the impurities include but are not limited to proteinsand related materials derived from cell lysates that are able to passthrough the column matrix. In some embodiments, the cell lysatederivatized product requires secondary purification to remove traceimpurities derived from cell lysates. In some embodiments, secondarypurification is any one of the methods disclosed herein for additionalpurification.

A column matrix is used herein to immobilize the heterogeneous metalcatalyst. Any type of solid support capable of immobilizing theheterogeneous metal catalyst is suitable for use as the column matrix.Suitable column matrices include but are not limited to, resins orsupports that are polystyrene-based, polysaccharide based,polyamide-based, carbon-based, alumina-based, and silica-based. Furtherexamples also include resins that are used for ion-exchangechromatography, affinity chromatography and size exclusionchromatography.

In some embodiments, the column matrix comprises a heterogeneous metalcatalyst. In some embodiments, the column matrix is suitable forcatalyzing the azide-alkyne cycloaddition. In some embodiments, thecolumn matrix comprises a heterogeneous metal catalyst that catalyzesthe azide-alkyne cycloaddition. In some embodiments, the heterogeneousmetal catalyst comprises a copper catalyst, a ruthenium catalyst, asilver catalyst, or a zinc catalyst. In some embodiments, theheterogeneous metal catalyst comprises a copper catalyst. In someembodiments, the heterogeneous metal catalyst comprises a rutheniumcatalyst. In some embodiments, the heterogeneous metal catalystcomprises a silver catalyst. In some embodiments, the heterogeneousmetal catalyst comprises a zinc catalyst.

In some embodiments, the column matrix further comprises a resin orsupport that is polystyrene-based, polysaccharide based,polyamide-based, carbon-based, alumina-based, silica-based, or anycombination thereof. In some embodiments, the column matrix furthercomprises a resin or support that is suitable for ion-exchangechromatography, affinity chromatography, or size exclusionchromatography. In some embodiments, the column matrix further comprisesan ion-exchange resin, an affinity resin, a size-exclusion or anycombination thereof.

In some embodiments, the column matrix further comprises an ion-exchangeresin. In some embodiments, ion-exchange resin is anion exchange resin.Examples of anion exchange resins include strong anion resins, such asthose containing quaternary ammonium groups, or weak anion exchangeresins, such as those containing ammonium chloride or hydroxide groups.

In some embodiments, the ion-exchange resin is a cation exchange resin.Examples of cation exchange resins include strong cation exchangeresins, such as those containing sulfonic acid groups or correspondingsalts, and weak cation exchange resins, such as those containingcarboxylic acid groups or the corresponding salts.

Examples of suitable ion-change resins include polystyrene-based resins,such as Amberlite®, Amberlyst®, Dowex®, Merrifield's peptide resin;polysaccharide-based resins, such as Sephadex®; polyethylenimine-basedresins; and polyamide-based resins.

In some embodiments, the ion-exchange resin is polystyrene-based. Insome embodiments, ion-exchange resin is polysaccharide-based. In someembodiments, the ion-exchange resin is polyethylenimine-based. In someembodiments, the ion-exchange resin is polyamide-based.

In some embodiments, the ion-exchange resin comprises polysaccharidesthat have been chemically modified. In some embodiments, theion-exchange resin comprises polysaccharides modified with carboxymethylfunctional groups, such as CM-cellulose. In some embodiments, theion-exchange resin comprises polysaccharides with diethylaminoethylgroups, such as DEAE-cellulose.

In some embodiments, the column matrix further comprises an additionalcolumn matrix. In some embodiments, the additional column matrixcomprises any type of solid support suitable for further purification.Suitable additional column matrices include but are not limited to,resins or supports that are polystyrene-based, polysaccharide based,polyamide-based, carbon-based, alumina-based, and silica-based. In someembodiments, the additional column matrix comprises a resin or supportthat is polystyrene-based, polysaccharide-based, polyamide-based,carbon-based, alumina-based, silica-based, or any combination thereof.In some embodiments, the additional column matrix comprises a resin orsupport that is polystyrene based. In some embodiments, the additionalcolumn matrix comprises a resin or support that is polysaccharide based.In some embodiments, the additional column matrix comprises a resin orsupport that is polyamide-based. In some embodiments, the additionalcolumn matrix comprises a resin or support that is carbon-based. In someembodiments, the additional column matrix comprises a resin or supportthat is alumina-based. In some embodiments, the additional column matrixcomprises a resin or support that is silica based.

Further examples also include resins that are used for ion-exchangechromatography, affinity chromatography, and size-exclusionchromatography. In some embodiments, the additional column matrixcomprises an ion-exchange resin, an affinity resin, a size-exclusion orany combination thereof. In some embodiments, the additional columnmatrix comprises a size-exclusion resin. In some embodiments, theadditional column matrix comprises an ion-exchange resin. In someembodiments, the additional column matrix comprises an affinity resin.Transition Metal Catalysts

In some embodiments, the column matrix comprises a heterogeneous metalcatalyst. In some embodiments, the heterogeneous metal catalyst is acatalyst suitable for catalyzing the azide-alkyne cycloaddition.Examples such heterogeneous metal catalysts include but are not limitedto copper catalysts, ruthenium catalysts, silver catalysts and zinccatalysts. In some embodiments, the heterogeneous metal catalystcomprises a copper catalyst, a ruthenium catalyst, a silver catalyst, ora zinc catalyst. In some embodiments, the heterogeneous metal catalystcomprises a copper catalyst. In some embodiments, the heterogeneousmetal catalyst comprises a ruthenium catalyst. In some embodiments, theheterogeneous metal catalyst comprises a silver catalyst. In someembodiments, the heterogeneous metal catalyst comprises a zinc catalyst.

In some embodiments, the heterogeneous metal catalyst further comprisesa heterogeneous metal precatalyst and reducing agent, wherein reductionof the precatalyst by the reducing agent generates the catalyticallyactive species. In some embodiments, the heterogeneous metal catalystfurther comprises a heterogeneous precatalyst and an oxidizing agent,wherein the oxidation of the precatalyst by the oxidizing agentgenerates the catalytically active species.

In some embodiments, the column matrix comprises a heterogeneousruthenium catalyst. In some embodiments, the heterogeneous rutheniumcatalyst comprises a ruthenium (II) catalyst. In some embodiments, theruthenium catalyst (II) further comprises a pentamethylcyclopentadienyl(Cp*) ligand. In some embodiments, the ruthenium catalyst (II) furthercomprises a cyclopentadienyl (Cp) ligand. In some embodiments, theruthenium catalyst comprises a pentamethylcyclopentadienyl rutheniumchloride [Cp*RuCl] complex. In some embodiments, the ruthenium catalystis pentamethylcyclopentadienylbis(triphenylphosphine)ruthenium (II)chloride (Cp*RuCl(PPh₃)₂). In some embodiments, the ruthenium catalystis chloro(1,5-cyclooctadiene)(pentamethylcyclopentadienyl)ruthenium(Cp*RuCl(COD), COD=1,5-cyclooctadiene).

In some embodiments, the column matrix comprises a heterogeneous silvercatalyst. In some embodiments, the heterogeneous silver catalystcomprises a silver (I) catalyst. In some embodiments, the column matrixcomprises a heterogeneous zinc catalyst.

Heterogeneous Copper Catalyst

In some embodiments, the column matrix comprises a heterogeneous coppercatalyst. In some embodiments, the heterogeneous copper catalystcomprises a copper(II) precatalyst and a reducing agent, wherein thereducing agent reduces the copper(II) precatalyst to form the activecopper (I) catalyst required for the azide-alkyne cycloaddition. In someembodiments, the heterogeneous copper catalyst comprises a copper (I)catalyst. In some embodiments, the heterogeneous copper catalystcomprises a copper(0) precatalyst and an oxidizing agent, wherein theoxidizing agent oxidizes the copper(0) precatalyst to form the activecopper (I) catalyst required for the azide-alkyne cycloaddition.

Copper (0) precatalysts contemplated for use include any copper (0)sources are capable of being oxidized to copper (I) by an oxidizingagent to catalyzing the azide-alkyne cycloaddition reaction. Examples ofsuch copper (0) precatalyst include but are not limited to copper metal,copper wire, copper turnings, copper powder and copper nanoparticles. Anexample of suitable oxidizing agent includes but is not limited tocopper sulfate.

Copper (I) catalysts contemplated for use include any copper (I) saltsor copper (I)-ligand that are capable of catalyzing the azide-alkynecycloaddition reaction. Examples of such copper (I) salts that aresuitable include, but are not limited, to copper(I) bromide, copper (I)iodide, and copper (I) tetrakis(acetonitrile) hexafluorophosphate.Examples of copper (I)-ligand complexes that are suitable include butnot limited to any copper (I)-ligand complexes wherein the ligand is anyone of the ligands described in this application.

Copper (II) precatalysts contemplated for use include any copper (II)salts or copper (II)-ligand complexes that are reduced to form thecatalytically active copper(I) species required for the copper catalyzedazide-alkyne cycloaddition reaction. Examples of such copper (II) saltsthat are suitable as copper (II) precatalysts include, but are notlimited, to copper(II) acetate, copper (II) chloride, copper (II)bromide, copper(II) trifluoromethanesulfonate, and copper (II) sulfate.Examples of copper (II)-ligand complexes that are suitable copper (II)precatalysts include but not limited, to any of the copper (II)-ligandcomplexes wherein the ligand is any of the ligands described in thisapplication.

In some embodiments, the copper (II) precatalyst is[Cu(1,10-phenanthroline-5,6-dione)₂]²⁺. In some embodiments, the copper(II) precatalyst is [Cu(4,7-diphenyl-1,10-phenanthroline-disulfonicacid)₂]²⁻.

In some embodiments, a ligand is employed to stabilize the active copper(I) catalyst. In some embodiments, the copper (II) precatalyst furthercomprises a ligand.

Examples of suitable ligands include, but are not limited to,substituted tris(triazolyl)methylamines andtris(benzimidazolylmethyl)amines. Examples oftris(triazolylmethyl)amines and tris(benzimidazolylmethyl)aminesinclude, but are not limited to,tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA);tris(3-hydroxypropyltriazolylmethyl)amine (THPTA);tris(2-benzimidazolylmethyl)amine (BimH)₃; and tripotassium5,5′,5″-[2,2′,2″-nitrilotris(methylene)tris(1H-benzimidazole-2,1-diyl)]tripentanoatehydrate (BimC4A)₃.

Examples of other ligands contemplated for use further include, but arenot limited to, bipyridines, phenanthrolines, pyridine bis(oxazoline)(PyBOX) and derivatives thereof. Examples of bipyridines andphenanthrolines include but are not limited to 2,2′-bipyridine,4-4′-dimethoxy-2-2′-bipyridine, 4,4′-dimethyl-2,2′-bipyridine,2,6-bis(2-pyridyl)pyridine (terpyridine),1,10-phenanthroline,bathophenanthroline, and 4,7-diphenyl-1,10-phenanthrolinedisulfonic acid(4,7-diphenyl-1,10-phenanthroline disulfonic acid disodium salthydrate).

In some embodiments, the ligand is phenanthroline derivative. In someembodiments, the ligand is 1,10-phenanthroline. In some embodiments, theligand is 4,7-diphenyl-1,10-phenanthrolinedisulfonic acid.

Reducing agents contemplated for use include any reducing agent capableof reducing the copper (II) precatalyst to form the catalytically activecopper (I) species required for the copper catalyzed azide-alkynecycloaddition. Suitable reducing agents include metallic based reducingagents, such as Cu, Al, Be, Co, Cr, Fe, Mg, Mn, Ni and Zn. A particularexample of a suitable reducing agent is zinc amalgam. Other reducingagents, include but are not limited to, ascorbate, quinone,hydroquinone, vitamin K₁, glutathione, and cysteine.

In some embodiments, the reducing agent is a metallic based reducingagent. In some embodiments, the reducing agent is zinc amalgam.

In some embodiments, further purification is achieved by incorporatingan additional matrix to the centrifuge column such that once thetriazole product has been formed in matrix comprising that heterogeneouscopper catalyst, the product is eluted through the additional matrix. Insome embodiments, the additional matrix is a resin that is suitable forthe purification of the triazole product. In some embodiments, theadditional matrix comprises a resin or support that ispolystyrene-based, polysaccharide-based, polyamide-based, carbon-based,alumina-based, silica-based, or any combination thereof. In someembodiments, the additional matrix comprises an ion-exchange resin, anaffinity resin, a size-exclusion or any combination thereof. In someembodiments the additional matrix is a size-exclusion resin.

In some embodiments, further purification is achieved by incorporating asize-exclusion resin to the centrifuge column such that once thetriazole product has been formed in matrix comprising that heterogeneouscopper catalyst, the product is eluted through a layer of size-exclusionresin. In some embodiments, the centrifuge column further comprises asize-exclusion resin. Size-exclusion resins suitable for use includethose that are polystyrene-based, polysaccharide-based, orpolyamide-based. In some embodiments, the size-exclusion resin ispolystyrene-based, polysaccharide-based, polyamide-based, or anycombination thereof. In some embodiments, the size-exclusion resin ispolystyrene-based. In some embodiments, the size-exclusion resin ispolysaccharide-based. In other embodiments, the size-exclusion resin ispolyamide based.

Column Matrix

Provided herein is a column matrix comprising a heterogeneous metalcatalyst. Also provided herein is a column matrix comprising aheterogeneous metal catalyst that catalysts the azide-alkynecycloaddition. In some embodiments, the heterogeneous metal catalystcomprises a copper catalyst, a ruthenium catalyst, a silver catalyst, ora zinc catalyst. In some embodiments, the column matrix comprises acopper catalyst. In some embodiments, the column matrix comprises aruthenium catalyst. In some embodiments, the column matrix comprises asilver catalyst. In some embodiments, the column matrix comprises a zinccatalyst. In some embodiments, the heterogeneous metal catalyst furthercomprises a ligand. In some embodiments, the column matrix comprises aheterogeneous metal precatalyst and reducing agent. In some embodiments,the column matrix comprises a heterogeneous metal precatalyst andoxidizing agent.

In some embodiments, the copper catalyst further comprises a ligand. Insome embodiments, the copper catalyst comprises a copper (I) catalyst.In some embodiments, the copper catalyst comprises a copper (II)precatalyst and a reducing agent. In some embodiments, the copper (II)precatalyst further comprises a ligand. In some embodiments, thereducing agent is zinc amalgam. In some embodiments, the copper catalystcomprises a copper (0) precatalyst and an oxidizing agent.

In some embodiments, the copper catalyst comprises a copper (II)precatalyst and a reducing agent, wherein the reducing agent reduces thecopper(II) precatalyst to form the active copper (I) catalyst requiredfor the azide-alkyne cycloaddition. In some embodiments, the coppercatalyst comprises a copper (I) catalyst. In some embodiments, thecopper catalyst comprises a copper(0) precatalyst and an oxidizingagent, wherein the oxidizing agent oxidizes the copper(0) precatalyst toform the active copper (I) catalyst required for the azide-alkynecycloaddition.

In some embodiments, the ruthenium catalyst further comprises a ligand.In some embodiments, the ruthenium catalyst comprises a ruthenium (II)catalyst.

Provided herein is a column matrix comprising a heterogeneous coppercatalyst. Also provided herein is a column matrix comprising aheterogeneous copper catalyst that catalysts the azide-alkynecycloaddition.

In some embodiments, the heterogeneous copper catalyst further comprisesa ligand. In some embodiments, the heterogeneous copper catalystcomprises a copper (I) catalyst. In some embodiments, the heterogeneouscopper catalyst comprises a copper (II) precatalyst and a reducingagent. In some embodiments, the copper (II) precatalyst furthercomprises a ligand. In some embodiments, the reducing agent is zincamalgam. In some embodiments, the heterogeneous copper catalystcomprises a copper (0) precatalyst and an oxidizing agent.

In some embodiments, the heterogeneous copper catalyst comprises acopper (II) precatalyst and a reducing agent, wherein the reducing agentreduces the copper(II) precatalyst to form the active copper (I)catalyst required for the azide-alkyne cycloaddition. In someembodiments, the heterogeneous copper catalyst comprises a copper (I)catalyst. In some embodiments, the heterogeneous copper catalystcomprises a copper(0) precatalyst and an oxidizing agent, wherein theoxidizing agent oxidizes the copper(0) precatalyst to form the activecopper (I) catalyst required for the azide-alkyne cycloaddition.

Provided herein is a column matrix comprising a copper (I) catalyst.Also provided herein is a column matrix comprising a copper (II)catalyst and a reducing agent. In some embodiments, the reducing agentis zinc amalgam. Further provided herein is a column matrix comprising acopper (II) catalyst and zinc amalgam.

Provided herein is a column matrix for use in bioconjugation comprisinga copper (I) catalyst. Also provided herein is a column matrix for usein bioconjugation comprising a copper (II) precatalyst and a reducingagent. In some embodiments, the reducing agent is zinc amalgam. Furtherprovided herein is a column matrix for use in bioconjugation comprisinga copper (II) precatalyst and zinc amalgam.

Provided herein is a column matrix for use in bioconjugation catalyzedby a heterogeneous copper catalyst comprising a copper (I) catalyst.Also provided herein is a column matrix for use in bioconjugationcatalyzed by a heterogeneous copper comprising a copper (II) precatalystand a reducing agent. In some embodiments, the reducing agent is zincamalgam. Further provided herein is a column matrix for use inbioconjugation catalyzed by a heterogeneous copper comprising a copper(II) precatalyst and zinc amalgam. In some embodiments, the copper (II)precatalyst further comprises a ligand.

In some embodiments, the column matrix further comprises a resin orsupport that is polystyrene-based, polysaccharide based,polyamide-based, carbon-based, alumina-based, silica-based, or anycombination thereof. In some embodiments, the column matrix furthercomprises a resin or support that is suitable for ion-exchangechromatography, affinity chromatography, or size exclusionchromatography. In some embodiments, the column matrix further comprisesan ion-exchange resin, an affinity resin, a size-exclusion or anycombination thereof.

In some embodiments, the column matrix further comprises an ion-exchangeresin. In some embodiments, the ion-exchange resin is an anion exchangeresin or a cation exchange resin. In some embodiments, the column matrixfurther comprises a size-exclusion resin.

In some embodiments, the column matrix is suitable for use in gravitycolumn chromatography, or in centrifugal column chromatography. In someembodiments, the column matrix is suitable use in gravity columnchromatography. In some embodiments, the column matrix is suitable foruse in centrifugal column chromatography. In some embodiments, thecentrifugal column chromatography is microspin column chromatography. Insome embodiments, column matrix is suitable for use in microspin columnchromatography. In some embodiments, the column matrix is suitable foruse in azide-alkyne cycloaddition.

In some embodiments, the column matrix further comprises an additionalmatrix. In certain embodiments, the additional matrix is a resinsuitable for the purification of a cycloaddition compound formed fromthe azide-alkyne cycloaddition (e.g., a triazole). In certainembodiments, the additional matrix is a resin suitable for thepurification of a product. In certain embodiments, the product is atriazole formed from a heterogeneous metal-catalyzed azide alkynecycloaddition. In some embodiments, the column matrix further comprisesan additional matrix that is a resin suitable for the purification of acycloaddition compound formed from the azide-alkyne cycloaddition. Incertain embodiments, the additional matrix is a size-exclusion resin.

The column matrix comprising the heterogeneous metal catalyst isprepared through a variety of methods. In some embodiments, the columnmatrix is prepared by mixing the metal catalyst with the appropriateresin or solid support. In some embodiments, the column matrix isprepared by adding the metal catalyst to a mixture comprising at leastone appropriate resin or solid support. In some embodiments, whenheterogeneous metal precatalysts are used, the column matrix is preparedby pre-mixing the metal precatalyst and resin or resins in anappropriate buffer solution or solvent to form a suspension and thenadding the appropriate reducing agent or oxidizing agent to thesuspension. The reducing agent or oxidizing agent is then mixed into thesuspension.

In some embodiments, the resin premixed with the metal catalyst ispolystyrene-based, polysaccharide-based, polyamide-based, carbon-based,alumina-based, silica-based, or any combination thereof. In someembodiments, the resin premixed with the metal catalyst ispolystyrene-based. In some embodiments, the resin premixed with themetal catalyst is polysaccharide-based. In some embodiments, the resinpremixed with the metal catalyst is polyamide-based. In someembodiments, the resin premixed with the metal catalyst is carbon-based.In some embodiments, the resin premixed with the metal catalyst isalumina-based. In some embodiments, the resin premixed with the metalcatalyst is silica-based.

In some embodiments, the resin premixed with the metal catalyst is anion-exchange resin, affinity resin, size-exclusion resin, or acombination thereof. In some embodiments, the resin premixed with themetal catalyst is an affinity resin. In some embodiments, the resinpremixed with the metal catalyst is a size-exclusion resin. In someembodiments, the resin premixed with the metal catalyst is anion-exchange. In other embodiments, the ion-exchange resin is a cationexchange resin. The type of ion-exchange resin used would depend on theoverall charge of the alkyne and/or azide components used. For instance,if an alkynyl DNA is used, then a cation exchange resin is used. If analkynyl peptide is positively charged, then an anion exchange column isused.

In some embodiments, the resin is premixed with a metal catalyst thatcatalyzes the azide-alkynyl cycloaddition. In some embodiments, theresin is premixed with a copper catalyst. In some embodiments, the resinis premixed with a ruthenium catalyst. In some embodiments, the resin ispremixed with a silver catalyst. In some embodiments, the resin ispremixed with a zinc catalyst. In some embodiments, the resin ispremixed with a metal precatalyst and reducing agent. In someembodiments, the resin is premixed a metal precatalyst and an oxidizingagent.

The column matrix comprising the heterogeneous copper catalyst isprepared similarly. In some embodiments, the column matrix is preparedby mixing the copper catalyst with the appropriate resin or solidsupport. In some embodiments, the column matrix is prepared by mixingthe copper catalyst to a mixture comprising at least one the appropriateresin or solid support. In some embodiments, the column matrix isprepared by pre-mixing the copper (II) precatalyst and resin in anappropriate buffer to form a suspension and then adding the reducingagent to the suspension. The reducing agent is then mixed into thesuspension. In some embodiments, the reducing agent added is zincamalgam. The column matrix will turn into an intense olive green,indicating that the catalytically active copper (I) catalyst has beenformed. The column matrix is then ready for use and is transferred tothe centrifuge column. In some embodiments, the reducing agent ispre-mixed with the resin or resins and the copper (II) precatalyst isadded to the pre-mixture. In some embodiments, a copper (I) catalyst isadded to the resin instead of a copper (II) precatalyst and a reducingagent. In some embodiments, a copper (0) precatalyst and oxidizing agentis used instead of a copper (II) precatalyst and a reducing agent.

In some embodiments, the resin premixed with the copper catalyst ispolystyrene-based, polysaccharide-based, polyamide-based, carbon-based,alumina-based, silica-based, or any combination thereof. In someembodiments, the resin premixed with the copper catalyst ispolystyrene-based. In some embodiments, the resin premixed with thecopper catalyst is polysaccharide-based. In some embodiments, the resinpremixed with the copper catalyst is polyamide-based. In someembodiments, the resin premixed with the copper catalyst iscarbon-based. In some embodiments, the resin premixed with the coppercatalyst is alumina-based. In some embodiments, the resin premixed withthe copper catalyst is silica-based.

In some embodiments, the resin premixed with the copper catalyst is anion-exchange resin, affinity resin, size-exclusion resin, or acombination thereof. In some embodiments, the resin premixed with thecopper catalyst is an affinity resin. In some embodiments, the resinpremixed with the copper catalyst is a size-exclusion resin. In someembodiments, the resin premixed with the copper catalyst is anion-exchange. In other embodiments, the ion-exchange resin is a cationexchange resin.

The type of ion-exchange resin used depends on the overall charge of thealkyne and/or azide components used. For instance, if an alkynyl DNA isused, then a cation exchange resin is used. If an alkynyl peptide ispositively charged, then an anion exchange column is used.

Furthermore, the type of cation-exchange resin used also correlates tothe type of copper (II) precatalyst used. In some embodiments, when acation-exchange resin is used, then the copper (II) precatalyst is[Cu(1,10-phenanthroline-5,6-dione)₂]²+. In some embodiments, when ananion-exchange resin is used, then the copper (II) precatalyst is[Cu(4,7-diphenyl-1,10-phenanthroline-disulfonic acid)2]²-.

Column

Provided herein is a column comprising a matrix of a heterogeneous metalcatalyst. In some embodiments, the heterogeneous metal catalystcomprises a metal catalyst suitable for catalyzing the azide-alkynecycloaddition. Also provided herein is a column comprising a matrix of aheterogeneous metal catalyst that catalyzes the azide-alkynecycloaddition. In some embodiments, the heterogeneous metal catalystcomprises a copper catalyst, a ruthenium catalyst, a silver catalyst, orzinc catalyst. In some embodiments, the heterogeneous metal catalystcomprises a copper catalyst. In some embodiments, the heterogeneousmetal catalyst comprises a ruthenium catalyst. In some embodiments, theheterogeneous metal catalyst comprises a silver catalyst. In someembodiments, the heterogeneous metal catalyst comprises a zinc catalyst.In some embodiments, the heterogeneous metal catalyst further comprisesa ligand. In some embodiments, the heterogeneous metal catalyst furthercomprises a heterogeneous metal precatalyst and reducing agent. In someembodiments, the heterogeneous metal catalyst further comprises aheterogeneous metal precatalyst and oxidizing agent.

In some embodiments, the copper catalyst further comprises a ligand. Insome embodiments, the copper catalyst comprises a copper (I) catalyst.In some embodiments, the copper catalyst comprises a copper (II)precatalyst and a reducing agent. In some embodiments, the copper (II)precatalyst further comprises a ligand. In some embodiments, thereducing agent is zinc amalgam. In some embodiments, the copper catalystcomprises a copper (0) precatalyst and an oxidizing agent.

In some embodiments, the copper catalyst comprises a copper (II)precatalyst and a reducing agent, wherein the reducing agent reduces thecopper (II) precatalyst to form the active copper (I) catalyst requiredfor the azide-alkyne cycloaddition. In some embodiments, the coppercatalyst comprises a copper (I) catalyst. In some embodiments, thecopper catalyst comprises a copper (0) precatalyst and an oxidizingagent, wherein the oxidizing agent oxidizes the copper (0) precatalystto form the active copper (I) catalyst required for the azide-alkynecycloaddition.

In some embodiments, the ruthenium catalyst further comprises a ligand.In some embodiments, the ruthenium catalyst comprises a ruthenium (II)catalyst.

Provided herein is a column comprising a matrix of a heterogeneouscopper catalyst. Also provided herein is a column comprising a matrix ofa heterogeneous copper catalyst that catalyzes the azide-alkynecycloaddition.

In some embodiments, the heterogeneous copper catalyst further comprisesa ligand. In some embodiments, the heterogeneous copper catalystcomprises a copper (I) catalyst. In some embodiments, the heterogeneouscopper catalyst comprises a copper (II) precatalyst and a reducingagent. In some embodiments, the heterogeneous copper (II) precatalystfurther comprises a ligand. In some embodiments, the reducing agent iszinc amalgam. In some embodiments, the heterogeneous copper catalystcomprises a copper (0) precatalyst and an oxidizing agent.

Provided herein is a column comprising a matrix of a copper (I)catalyst. Also provided herein is a column comprising a matrix of acopper (II) precatalyst and a reducing agent. In some embodiments, thereducing agent is zinc amalgam. Further provided is a column comprisinga matrix of a copper (II) precatalyst and zinc amalgam.

Provided herein is a column for use in bioconjugation comprising amatrix of a copper (I) catalyst. Also provided herein is a column foruse in bioconjugation comprising a matrix of a copper (II) precatalystand a reducing agent. In some embodiments, the reducing agent is zincamalgam. Further provided is a column for use in bioconjugationcomprising a matrix of a copper (II) precatalyst and zinc amalgam.

Provided herein is a column for use in bioconjugation catalyzed by aheterogeneous copper catalyst comprising a matrix of a copper (I)catalyst. Also provided herein is a column for use in bioconjugationcatalyzed by a heterogeneous copper catalyst comprising a matrix of acopper (II) precatalyst and a reducing agent. In some embodiments, thereducing agent is zinc amalgam. Further provided is a column for use inbioconjugation catalyzed by a heterogeneous copper catalyst comprising amatrix of a copper (II) precatalyst and zinc amalgam.

In some embodiments, the column is a gravity column or a centrifugecolumn. In some embodiments, the column is a gravity column. In someembodiments, the column is a centrifuge column. In some embodiments, thecentrifuge column is suitable for use in microspin columnchromatography.

A schematic showing an embodiment of the column is shown in FIG. 1. Asused herein, a column is meant to encompass any column that is suitablefor any sample size and volume as long as the column allows forchromatographic separation. As shown in FIG. 1, the matrix comprisingthe heterogeneous metal catalyst is loaded into the column. Furthermore,in some embodiments the column comprises an additional column matrix toallow for further purification.

In some embodiments, the column further comprises an additional columnmatrix. In some embodiments, the column further comprises at least oneadditional column matrix. In some embodiments, the additional columnmatrix is a resin suitable for the purification of a cycloadditioncompound formed from the azide-alkyne cycloaddition. In certainembodiments, the additional column matrix is a resin suitable for thepurification of a product. In certain embodiments, the product is atriazole formed from a heterogeneous metal-catalyzed azide alkynecycloaddition.

In some embodiments, the column further comprises an additional columnmatrix that is a resin suitable for the purification of a cycloadditioncompound formed from the azide-alkyne cycloaddition.

Such additional columns matrices are resins that include and are notlimited to affinity column resins, ion-exchange resins, orsize-exclusion resins. In some embodiments, additional column matrix isan affinity resin, an ion-exchange resin, or size-exclusion resin. Insome embodiments, the column further comprises an affinity column resin,an ion-exchange resin, a size-exclusion resin or any combinationthereof.

In some embodiments, the additional column matrix is polystyrene-based,polysaccharide-based, polyamide-based, carbon-based, alumina-based,silica-based, or any combination thereof. In some embodiments, theadditional column matrix is polystyrene-based. In some embodiments, theadditional column matrix is polysaccharide-based. In some embodiments,the additional column matrix is polyamide-based. In some embodiments,the additional column matrix is carbon-based. In some embodiments, theadditional column matrix is alumina-based. In some embodiments, theadditional column matrix is silica-based.

Although FIG. 1 shows a column comprising of at least one additionalcolumn matrix, there are embodiments wherein a column has two, three,four, or more additional column matrices. In some embodiments, theadditional column matrix or matrices precede the matrix comprising theheterogeneous metal complex catalysts. In some embodiments, theadditional column matrices precede and follow the matrix comprising theheterogeneous catalyst.

Furthermore, although FIG. 1 is a schematic for an embodiment of acentrifuge column, that configuration of the column shown in FIG. 1 isnon-limiting. The columns contemplated for use are any columns capableof chromatographic separation for any sample size and volume. Suchcolumns include columns wherein the solvent or buffer solution is passedthrough the column matrix by gravity or centrifugal force. In someembodiments, wherein the solvent or buffer solution is passed throughthe column matrix through gravity, positive pressure from air orcompressed gas is also used to facilitate solvent flow. Examples ofsuitable compressed gas include compressed air, nitrogen, and argon.

In some embodiments, the column is suitable for use in gravitychromatography and centrifugal chromatography. In some embodiments, thecolumn is suitable for gravity chromatography. In some embodiments, thecolumn is suitable for use in centrifugal chromatography.

Provided herein is a gravity column comprising a matrix of a copper (I)catalyst. Also provided herein is a gravity column comprising a matrixof a copper (II) precatalyst and a reducing agent. In some embodiments,the reducing agent is zinc amalgam. Further provided is a gravity columncomprising a matrix of a copper (II) precatalyst and zinc amalgam.

Provided herein is a gravity column for use in bioconjugation comprisinga matrix of a copper (I) catalyst. Also provided herein is a gravitycolumn for use in bioconjugation comprising a matrix of a copper (II)precatalyst and a reducing agent. In some embodiments, the reducingagent is zinc amalgam. Further provided is a gravity column for use inbioconjugation comprising a matrix of a copper (II) precatalyst and zincamalgam.

Provided herein is a gravity column for use in bioconjugation catalyzedby a heterogeneous copper catalyst comprising a matrix of a copper (I)catalyst. Also provided herein is a gravity column for use inbioconjugation catalyzed by a heterogeneous copper catalyst comprising amatrix of a copper (II) precatalyst and a reducing agent. In someembodiments, the reducing agent is zinc amalgam. Further provided is agravity column for use in bioconjugation catalyzed by a heterogeneouscopper catalyst comprising a matrix of a copper (II) precatalyst andzinc amalgam.

Provided herein is a centrifuge column comprising a matrix of a copper(I) catalyst. Also provided herein is a centrifuge column comprising amatrix of a copper (II) precatalyst and a reducing agent. In someembodiments, the reducing agent is zinc amalgam. Further provided is acentrifuge column comprising a matrix of a copper (II) precatalyst andzinc amalgam.

Provided herein is a centrifuge column for use in bioconjugationcomprising a matrix of a copper (I) catalyst. Also provided herein is acentrifuge column for use in bioconjugation comprising a matrix of acopper (II) precatalyst and a reducing agent. In some embodiments, thereducing agent is zinc amalgam. Further provided is a centrifuge columnfor use in bioconjugation comprising a matrix of a copper (II)precatalyst and zinc amalgam.

Provided herein is a centrifuge column for use in bioconjugationcatalyzed by a heterogeneous copper catalyst comprising a matrix of acopper (I) catalyst. Also provided herein is a centrifuge column for usein bioconjugation catalyzed by a heterogeneous copper catalystcomprising a matrix of a copper (II) precatalyst and a reducing agent.In some embodiments, the reducing agent is zinc amalgam. Furtherprovided is a centrifuge column for use in bioconjugation catalyzed by aheterogeneous copper catalyst comprising a matrix of a copper (II)precatalyst and zinc amalgam.

In some embodiments, the matrix further comprises an ion-exchange resin.In some embodiments, the ion-exchange resin is an anion exchange resinor a cation exchange resin. In some embodiments, the matrix furthercomprises a size-exclusion resin. In some embodiments, the copper (II)precatalyst further comprises a ligand. In some embodiments, thecentrifuge column is suitable for use in azide-alkyne cycloaddition.

A schematic showing an embodiment of the centrifuge column is shown inFIG. 2. As used herein, a centrifuge column is meant to encompass anycolumn that is suitable for any sample size and volume as long as thecolumn allows for centrifugation. In some embodiments, the centrifugecolumn is suitable for use in microspin column chromatography. As shownin FIG. 2, the matrix comprising the heterogeneous copper catalyst isloaded in to the centrifuge column. Furthermore, in some embodiments thecentrifuge column comprises an additional column matrix to allow forfurther purification. The additional column matrix shown in FIG. 2 is asize-exclusion resin.

In some embodiments, the centrifuge column further comprises at leastone additional column matrix. In certain embodiments, the additionalcolumn matrix is a resin suitable for the purification of a product. Incertain embodiments, the product is a triazole formed from aheterogeneous metal-catalyzed azide alkyne cycloaddition.

Such additional columns matrices are resins that include and are notlimited to affinity column resins, ion-exchange resins, orsize-exclusion resins. In some embodiments, additional column matrix isan affinity resin, an ion-exchange resin, or size-exclusion resin. Insome embodiments, the centrifuge column further comprises an affinitycolumn resin, an ion-exchange resin, a size-exclusion resin or anycombination thereof

In some embodiments, the additional column matrix is polystyrene-based,polysaccharide-based, polyamide-based, carbon-based, alumina-based,silica-based, or any combination thereof. In some embodiments, theadditional column matrix is polystyrene-based. In some embodiments, theadditional column matrix is polysaccharide-based. In some embodiments,the additional column matrix is polyamide-based. In some embodiments,the additional column matrix is carbon-based. In some embodiments, theadditional column matrix is alumina-based. In some embodiments, theadditional column matrix is silica-based.

Although FIG. 2 shows a centrifuge column comprising of at least oneadditional column matrix, there are embodiments wherein a centrifugecolumn has two, three, four, or more additional column matrices.

Furthermore, although FIG. 2 is a schematic for an embodiment of acentrifuge column, that configuration of the column shown in FIG. 2 isnon-limiting.

EXAMPLES

The present invention may be better understood through reference to thefollowing examples. These examples are included to describe exemplaryembodiments only and should not be interpreted to encompass the entirebreadth of the invention.

Example 1 Preparation of Zinc Amalgam

3 g of zinc granules (˜1 mm diameter) were placed in a 500 mL Erlenmeyerflask. The granules were washed with 2 mL of 6 M HCl for 5 seconds andthen washed 8 times with 10 mL aliquots of water. After the final wash,15 mL of 0.25 M HgCl₂ were added, and the flask was left to sit for 5minutes. The liquid was then poured off, and the zinc amalgam was washedwith 10 mL aliquots of water 8 more times.

Example 2 Preparation of Centrifuge Column with Matrix ComprisingHeterogeneous Copper Catalyst

A schematic of the one embodiment of the centrifuge column comprisingthe heterogeneous copper catalyst is shown in FIG. 2. The apparatusconsists of a modified Bio Rad microspin column (2 cm diameter)preloaded with ˜100 μL of 6 kD size-exclusion resin (Bio-Gel P-6 media).Small pellets (˜1 mm diameter) of amalgamated zinc were mixed into 400μL of an ion-exchange slurry in either ammonium acetate buffer (0.1 M,pH 7.5) or 0.1% aqueous trifluoroacetic acid, then layered on top of thecolumn. Next, 500 μL of a 10 mM solution of either Cu^(II)(phendione)₂²⁺ (for cation-exchange resins, Sephadex-CM C-50; phendione is1,10-phenanthroline-5,6-dione) or Cu^(II)(bathophen)₂ ²⁻ (foranion-exchange resins, Sephadex-DEAE A-50; bathophen is4,7-diphenyl-1,10-phenanthroline-disulfonic acid disodium salt) wereadded to the top of the column and immobilized onto the matrix byspinning at 1000 rpm for 1 minute in a microcentrifuge. The column waswashed several times with deoxygenated buffer, after which theion-exchange layer appeared as an intense olive green color, indicatingthe presence of catalytically active Cu^(I)(phendione)₂ ⁺ orCu^(I)(bathophen)₂ ⁻. Columns prepared in this fashion are optionallystored in the refrigerator for at least two months prior to use, with nodeterioration in their activity.

Example 3 Preparation of Glycosylated DNA Matrix Preparation

Approximately 250 μL of CM-52 cation-exchange resin was suspended in anequal volume of 50 mM ammonium acetate buffer pH 6.5. A separate 150 μLaliquot of CM-52 was similarly suspended in an aqueous solution (10 mM)of Cu(phendione)₂ ²⁺ that had been previously degassed with argon.Before preparing the column, a small amount of zinc amalgam (4 or 5grains) was added to the CM-52/Cu(phendione)₂ ²⁺ mixture and shakenuntil it turned dark green.

Centrifuge Column Preparation

The centrifuge column consisted of a modified Bio Rad MicrospinChromatography column. The commercial column was opened and the majorityof the pre-packed size-exclusion gel (P-6 polyacrylamide gel, sizeexclusion limit 6 kDa) was extracted, leaving only a small amount (˜100μL) suspended in tris buffer. A small amount (˜200 μL) of the pureion-exchange resin was layered on top, followed by another ˜200 μL ofthe ion-exchange resin containing Cu (I) and zinc amalgam. The resultingcolumn was washed 5 times with 100 μl of degassed 50 mM ammonium acetatebuffer by spinning the column for 2 minutes at 1000×g in amicrocentrifuge.

Heterogeneous Copper-Catalyzed Azide-Alkyne Cycloaddition

50 μl of 200 μM ethynyl-labeled DNA(5′-ethynyl-(CH₂)₆-GCTCAGTACGACGTCGA-3′; MW 5355.5 g/mol; 200 μM) wascombined with 50 μl of 1-azido-1-deoxy-β-D-galactopyranoside ranging inconcentration from 200 μM to 20 mM. This solution was added to a spincolumn prepared using the cation exchange resin as described above andspun for 3 minutes at 1000×g. The column was then washed with five 100μl aliquots of 50 mM ammonium acetate buffer, each time spinning for 2min at 1000×g. The washes were collected and combined with the initialreaction.

HPLC analysis was used to determine the purity of the glycosylated DNAprepared. For comparison, the same DNA glycosylation reaction wasperformed via a more conventional approach, using Cu(phendione)₂ ²⁺ (1mM) with sodium ascorbate (10 mM). HPLC analysis was carried out usingan Agilent Model 1100 HPLC equipped with a Varian Dynamax 250×10 mm C18column. DNA samples were eluted with a mixture of acetonitrile and 50 mMammonium acetate buffer. The initial gradient was 3% acetonitrile for 5minutes, ramped to a final concentration of 35% over 60 minutes.

The HPLC chromatogram for the centrifuge column, or spin column,reaction yielded the remarkably clean trace shown in FIG. 3A, indicatingthe presence of only the desired product in the solution. The yield wasdetermined by drying down the product collected from the HPLC, followedby re-suspending it in a known quantity of buffer. The yield of theglycosylated DNA was typically isolated in >85% yield. Thenear-quantitative yield and essentially pure product obtained via thecentrifuge column is in sharp contrast to the more conventionalsolution-phase reaction. FIG. 3B depicts the resulting HPLC chromatogramof the product from the conventional approach and the chromatogramclearly shows several species in the mixture, indicating that furtherpurification is necessary to isolate the product.

Products were confirmed by MALDI-MS analysis. 3-hydroxypicolinic acid(3-HPA) was used for the MALDI matrix, consisting of 70 mg 3-HPA with 7mg diammonium citrate in 1.5 mL 10:1 deionized water:acetonitrile. Thecrude glycosylated DNA products were dried under vacuum, desalted, andthen spotted onto the MALDI sample plate with an equi-volume amount ofthe prepared 3-HPA matrix.

Example 4 Preparation of Fluorescent Peptide Matrix Preparation

The anionic resin was prepared in a similar fashion as described for theMatrix Preparation Section of Example 3. In this preparation, DE-52anion-exchange resin was suspended in 0.1% trifluoroacetic acid (TFA) indeionized water and 10 mM aqueous Cu(bathophenanthroline)₂ ²⁻ was usedas the copper (II) precatalyst.

Centrifuge Column Preparation

The centrifuge column consisted of a modified Bio Rad MicrospinChromatography column. The commercial column was opened and the majorityof the pre-packed size-exclusion gel (P-6 polyacrylamide gel, sizeexclusion limit 6 kDa) was extracted, leaving only a small amount (˜100μL) suspended in tris buffer. A small amount (˜200 μL) of the pureion-exchange resin was layered on top, followed by another ˜200 μL ofthe ion-exchange resin containing Cu (I) and zinc amalgam. The resultingcolumn was washed 5 times with 100 μl of degassed 50 mM ammonium acetatebuffer by spinning the column for 2 minutes at 1000×gin amicrocentrifuge.

Heterogeneous Copper-Catalyzed Azide-Alkyne Cycloaddition

The centrifuge column was washed with 300 μl of degassed 1% TFA indeionized water. Next, 50 μl of 8 mM coumarin-N₃ (fluorogenic dye3-azido-7-hydroxycoumarin) in a 3:1 mixture of water:DMSO was combinedwith 50 μl of 8 mM 8-Arg (octa-arginine) and bubble degassed with argon.The 8-Arg/coumarin-N₃ mixture was loaded into the column and spun for 2minutes at 1000×g. The column was then washed with five 100 μl aliquotsof 1% TFA in distilled water. The washes were collected and combinedwith the initial reaction.

HPLC analysis was used to determine the purity of the coumarin-labeled8-Arg. For comparison, we also carried out the same cycloadditionreaction with conventional solution-phase conjugation using sodiumascorbate as the reductant. HPLC analysis was carried out using anAgilent Model 1100 HPLC equipped with a Varian Dynamax 250×10 mm C18column. 8-Arg samples were eluted using 0.1% TFA in acetonitrile as themobile phase and 0.1% TFA in deionized water as the aqueous phase.Initial conditions were 25% acetonitrile for 5 minutes ramped to 50%over 40 minutes.

FIG. 4A and FIG. 4B show the HPLC traces of the crude products obtainedfrom the centrifuge column (FIG. 4A) as well as the products obtainedvia conventional solution-phase conjugation using sodium ascorbate asthe reductant (FIG. 4B). As observed with the glycosylated DNA reactionfrom Example 3, the centrifuge column, or spin-column, method results inessentially quantitative yield of coumarin-labeled 8-Arg.

Products were confirmed by MALDI-MS analysis. 3-hydroxypicolinic acid(3-HPA) was used for the MALDI matrix, consisting of 70 mg 3-HPA with 7mg diammonium citrate in 1.5 mL 10:1 deionized water:acetonitrile. Thecrude coumarin-labeled 8-Arg products were dried under vacuum, desalted,and then spotted onto the MALDI sample plate with an equi-volume amountof the prepared 3-HPA matrix.

Example 5 ICP-AES Analysis of Centrifuge Columns ContainingHeterogeneous Copper Catalyst Matrix

The columns described herein are used for these studies consisted of amodified Micro Bio-Spin column preloaded with ˜100 μL Bio-Gel P-6 sizeexclusion media (See FIG. 2 as an example). Small pellets (˜1 mmdiameter) of amalgamated zinc were mixed into 400 μL of an ion-exchangeslurry in either ammonium acetate buffer (0.1 M, pH 7.5) or 0.1% aqueousTFA, then layered on top of the column. Next, 500 μL of a 10 mM solutionof either Cu^(II)(phendinone)₂ ²⁺ (for cation-exchange resins,Macro-Prep CM resin) or Cu^(II)(bathophen)₂ ²⁻ for anion-exchangeresins, Macro-Prep DEAE resin) were added to the top of the column andimmobilized onto the matrix by spinning at 1000×g for 1 min in amicrocentrifuge. The column was washed several times with deoxygenatedbuffer, after which the ion-exchange layer appeared as an intense olivegreen color, indicated the presence of a catalytically activeCu^(I)(phendione)₂ ⁺ or Cu^(I)(bathophen)₂ ³⁻. The columns prepared inthis fashion have been stored in the refrigerator for at least 2 monthsprior to use with no deterioration in their activity.

ICP-AES analysis was conducted with a Perkin Elmer Optima 7000 DV. Acopper stock solution was prepared by dissolving copper(II) sulfatehexahydrate in double-distilled water and preparing appropriatedilutions with volumetric flasks to generate 0, 0.01, 0.05, and 0.2 mg/Lstandards. Eluent samples from the column were analyzed withoutadditional preparation. For each standard and sample, the instrument wasconfigured to average the intensity readings from two measurements.

FIG. 5 depicts the calibration curve obtained from the ICP-AES analysisof Cu in spin column flow-through loaded with Cu-ligand catalyst. Table1 corresponds to the data obtained used to prepare the calibration curvedepicted in FIG. 5.

TABLE 1 mg/L Mean Corrected Intensity Std. Dev. 0 1374.7 0.52 0.0168877.7 61.05 0.05 329245.9 1411.51 0.2 1457178.3 26164.55

Table 2 depicts the amount of copper detected with each eluent sampleanalyzed (BP=. Cu(bathophen) and PD=Cu(phendione)). The calibration dataindicated that 0.01 ppm approached the lower detection limit of theinstrument. All samples but one (PD with 1 mM EDTA in eluent) were belowthe detection limit of the instrument. These results show that thecolumns show good stability even in the presence of strongly chelatingions as the ICP-AES analysis showed that there were no detectable copperions in the flow-through in elution solutions containing up to 1 mMEDTA.

TABLE 2 Entry Sample mg/L Cu [Cu] (μM) 1 BP water wash 0.003 0.0472098952 BP 0.001 mM EDTA 0.002 0.031473263 3 BP 1 mM EDTA 0.002 0.031473263 4PD water wash 0.002 0.031473263 5 PD 0.001 mM EDTA 0.003 0.047209895 6PD 1 mM EDTA 0.193 3.037169924

Example 6 General Preparation of Bioconjugated Cycloaddition Compoundsfrom Centrifuge Columns Containing Heterogeneous Copper Catalyst Matrix

Using any one of the columns described herein, such as those describedin the Examples section, the copper-catalyzed azide-alkyne reaction iscarried out by pipetting onto the column a mixture of the appropriatereaction partners, such as a alkyne-labeled biomolecule, in theappropriate concentration, such as from about 10 μm to about 10 mM, andan azide-labeled ligand in the appropriate concentration ratio, such asa ratio ranging from about stoichiometric to 100-fold excess. The columnis then subjected to spinning via a microcentrifuge for an appropriateamount of time, such as several minutes. The column is then washed withan appropriate solution followed by spinning for an appropriate amountof time to elute the product, such as the bioconjugated product.

In some embodiments, the mixture of appropriate reaction partnerscontain cells and/or cell lysates. In some instances, when the mixtureof the appropriate reaction partners is passed through the column, themajority of the cells and/or cell lysates remain at the top of thecolumn and do not pass through the column. In some instances, thebioconjugated product obtained is free or substantially free ofimpurities derived from the cells and/or cell lysates. In someinstances, the bioconjugated product obtained contains trace impuritiesderived from the cells and/or cell lysates and a second purification isrequired to remove the trace impurities.

In some instances, the columns are stored in the refrigerator for aperiod of time prior to use, such as for at least two months with nodeterioration in activity. In some instances, these columns are re-usedwithout any apparent loss in activity.

What is claimed is:
 1. A method for preparing a cycloaddition compoundfrom a reaction catalyzed by a heterogeneous copper catalyst, comprising(a) mixing an alkyne component and an azide component in an appropriatesolvent to form a solution; (b) transferring the solution containing thealkyne component and the azide component to a column with a matrixbarrier at the bottom of the column; wherein the column comprises amatrix of a copper (II) precatalyst and a reducing agent; wherein acopper (I) catalyst is generated from the reduction of the copper (II)precatalyst with the reducing agent; and (c) passing the solutioncontaining the alkyne component and the azide component through thecolumn; wherein upon contact with the copper (I) catalyst within thematrix, the alkyne component and the azide component react to form thecycloaddition compound.
 2. The method of claim 1, wherein the alkynecomponent comprises a small molecule, a protein, a peptide, an aminoacid, an oligonucleotide, a nucleotide, a nucleoside, a carbohydrate, ora fluorophore.
 3. The method of claim 1, wherein the azide componentcomprises a small molecule, a protein, a peptide, an amino acid, anoligonucleotide, a nucleotide, a nucleoside, a carbohydrate, or afluorophore.
 4. The method of claim 1, wherein the alkyne componentand/or the azide component contains cells and/or cell lysates.
 5. Themethod of claim 1, wherein the copper (II) precatalyst further comprisesa ligand.
 6. The method of claim 1, wherein the reducing agent is zincamalgam.
 7. The method of claim 1, wherein the matrix further comprisesan ion-exchange resin.
 8. The method of claim 1, wherein the methodfurther comprises passing the solution containing the cycloadditioncompound through an additional matrix.
 9. The method of claim 8, whereinthe additional matrix is a resin suitable for the purification of thecycloaddition compound.
 10. The method of claim 9, wherein theadditional matrix is a size-exclusion resin.
 11. The method of claim 1,wherein the column is suitable for use in gravity column chromatographyor centrifugal column chromatography.
 12. The method of claim 1, whereinthe cycloaddition compound is a cell lysate derivatized product.
 13. Themethod of claim 12, wherein the cell lysate derivatized product is freeor substantially free of impurities derived from cell lysates.
 14. Themethod of claim 12, wherein the cell lysate derivatized product requiresadditional purification to remove trace impurities derived from celllysates.
 15. A column matrix comprising a heterogeneous copper catalystthat catalyzes the azide-alkyne cycloaddition.
 16. The column matrix ofclaim 15, wherein the heterogeneous copper catalyst comprises a copper(II) precatalyst and a reducing agent.
 17. The column matrix of claim16, wherein the reducing agent is zinc amalgam.
 18. The column matrix ofclaim 15, wherein the column matrix further comprises an ion-exchangeresin.
 19. The column matrix of claim 15, wherein the column matrixfurther comprises an additional column matrix.
 20. The column matrix ofclaim 19, wherein the additional column matrix is a resin suitable forthe purification of a cycloaddition compound formed from theazide-alkyne cycloaddition.
 21. The column matrix of claim 20, whereinthe additional column matrix is a size-exclusion resin.
 22. The columnmatrix of claim 15, wherein the column matrix is suitable for use ingravity column chromatography or in centrifugal column chromatography.23. A column comprising a matrix of a heterogeneous copper catalyst thatcatalyzes the azide-alkyne cycloaddition.
 24. The column of claim 23,wherein the heterogeneous copper catalyst comprises a copper (II)precatalyst and a reducing agent.
 25. The column of claim 24, whereinthe reducing agent is zinc amalgam.
 26. The column of claim 23, whereinthe matrix further comprises an ion-exchange resin.
 27. The column ofclaim 23, wherein the column further comprises an additional matrix. 28.The column of claim 27, wherein the additional matrix is a resinsuitable for the purification of a cycloaddition compound formed fromthe azide-alkyne cycloaddition.
 29. The column of claim 28, wherein theadditional matrix is a size-exclusion resin.
 30. The column of claim 23,wherein the column is a gravity column or a centrifuge column.